Novel phospholipase D polypeptide and DNA sequences

ABSTRACT

Provided are novel phospholipase D DNA and amino acid sequences. The sequences are useful in methods and compositions for identifying phospholipase D mediator molecules which are in turn useful in therapeutic pharmacuetical compositions for treating rheumatoid arthritis, psoriasis, ulcerative colitis, in wound healing and for treating other diseases or conditions characterized by exhibition of an inflammatory response or in the treatment of cancer and other diseases characterized by pathogenic mitogenicity.

TECHNICAL FIELD

[0001] This invention is in the field of molecular biology andparticularly relates to nucleic acid sequences that encode novelphospholipases.

BACKGROUND

[0002] The mechanism by which specificity of physiological responses areconferred by a limited number of signal transducing substances,typically enzymes, is poorly understood. Cellular receptors on thesurfaces of various cells are involved and initiate multiple signalingpathways. Some of the receptors on neutrophils are known: the PAFreceptor, the interleukin-8 receptor and the fMetLeuPhe receptor allbelong to the super-family of G-protein-linked receptors. A commonfeature of these receptors is that they span the cell membrane seventimes, forming three extracellular and three intracellular loops and acytoplasmic carboxy-terminal tail. The third loop and the tail exhibitextensive variability in length and sequence, leading to speculationthat these parts are responsible for the selective interaction with thevarious G-proteins. Many of these G-protein-linked receptors stimulatethe activation of three phospholipases, phospholipase C (PLC),phospholipase D (PLD) and phospholipase A₂ (PLA₂). These phospholipasesconstitute a family of regulatory enzymes which trigger variousneutrophilic functions, for example adherence, aggregation, chemotaxis,exocytosis of secretory granules and activation of NADPH oxidase, i.e.,the respiratory burst.

[0003] The main substrates for the phospholipases are membranephospholipids. The primary substrates for PLC are the inositolcontaining lipids, specifically and typically phosphotidylinositol (PI).PI is phosphorylated by PLC resulting in the formation of PIP,phosphotidylinositol 4-phosphate. The primary substrate for PLD and PLA₂is phosphatidylcholine (PC), a relatively ubiquitous constituent of cellmembranes. The activity of cytosolic PLA₂ on PC liberates arachidonicacid, a precursor for the biosynthesis of prostaglandins andleukotrienes and possible intracellular secondary messenger. PLD, on theother hand, catalyzes the hydrolytic cleavage of the terminal phosphatediester bond of glycerophospholipids at the P-O position. PLD activitywas originally discovered in plants and only relatively recentlydiscovered in mammalian tissues. PLD has been the focus of recentattention due to the discovery of its activation by fMetLeuPhe inneutrophils. PLD activity has been detected in membranes and in cytosol.Although a 30 kD (kilodalton) and an 80 kD activity have been detected,it has been suggested that these molecular masses represented a singleenzyme with varying extents of aggregation. See Cockcroft, Biochimica etBiophysica Acta 1113: 135-160 (1992). One PLD has been isolated, clonedand partially characterized. See Hammond, J. Biol. Chem. 270:29640-43(1995). Biological characterization of PLD1 revealed that it could beactivated by a variety of G-protein regulators, specifically PKC(protein kinase C), ADP-ribosylation factor (ARF), RhoA, Rac1 andcdc-42, either individually or together in a synergistic manner,suggesting that a single PLD participates in regulated secretion incoordination with ARF and in propagating signal transduction responsesthrough interaction with PKC, PhoA and Rac1. Nonetheless,PKC-independent PLD activation has been associated with Src and Rasoncogenic transformation, leaving open the possibility that additionalPLDs might exist. See Jiang, Mol. and Cell. Biol. 14:3676 (1994) andMorris, Trends in Pharmacological Sciences 17: 182-85(1996). Thedifficulty may arise at least in part from the fact that in thephospholipase family enzymes may or may not be activated by, andcatalyze, multiple substances, making sorting, tracking andidentification by functional activities impractical.

[0004] There exists a need in the art for the identification andisolation of phospholipase enzymes. Without such identification andisolation, there is no practical way to develop assays for testingmodulation of enzymatic activity. The availability of such assaysprovides a powerful tool for the discovery of modulators ofphospholipase activity. Such modulators would be excellent candidatesfor therapeutics for the treatment of diseases and conditions involvingpathological mitogenic activity or inflammation.

SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides novel mammalianphospholipase D (PLD) proteins, which are substantially free from otherproteins with which they are typically found in their native state.These novel mammalian PLD proteins include polypeptides substantiallyfree of association with other polypeptides and comprising an enzyme ofmammalian origin having a phosphatidylcholine-specific pohspholipase Dactivity and containing at least two copies of the amino acid motifHXKXXXXD. More specifically, these proteins include polypeptidessubstantially free of association with other polypeptides and comprisingPLD polypeptides that are perinuclear membrane associated, requirePI(4,5)P2 for in vitro activity and are activated by one or moreG-proteins. Alternatively, these proteins include polypeptidessubstantially free of association with other polypeptides and comprisingPLD polypeptides that are plasma membrane associated, activatecytoskelatal reorganization pathways, require PI(4,5)P2 for in vitroactivity and do not require Rac1, cdc42, RhoA, PKC or ARF1 foractivation.

[0006] These novel mammalian PLD proteins may be produced by recombinantgenetic engineering techniques. They may also be purified from cellsources producing the enzymes naturally or upon induction with otherfactors. They may also be synthesized by chemical techniques, or acombination of the above-listed techniques. Mammalian PLD proteins fromseveral species, termed PLD1a, PLD1b and PLD2, have been isolated. HumanPLD1a and PLD1b are identical in amino acid sequence (SEQ ID NOS:2 and 5respectively) except for a 38 amino acid segment that is missing fromhPLD1b (SEQ ID NO:5), and present in hPLD1a (SEQ ID NO:2), beginning atamino acid number 585. Active mature PLD1a (SEQ ID NO:2) is anapproximately 1074 amino acid protein, characterized by an apparentmolecular weight for the mature protein of approximately 120 kD(kilodaltons) as determined by sodium dodecylsulfate polyacrylamide gelelectrophoresis of protein purified from baculovirus expressing cells.The calculated molecular weight for the mature protein is approximately124 kD. Active mature PLD1b (SEQ ID NO:5) is an approximately 1036 aminoacid protein, characterized by an apparent molecular weight ofapproximately 120 kD as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis of protein purified from baculovirusexpressing cells. The calculated molecular weight for the mature proteinis approximately 120 kD. Active mature PLD2 (SEQ ID NO:8) is anapproximately 932 amino acid protein, characterized by an apparentmolecular weight of approximately 112 kD as determined by sodiumdodecylsulfate polyacrylamide gel electrophoresis of protein purifiedfrom baculovirus expressing cells. The calculated molecular weight forthe mature protein is approximately 106 kD. As used herein, “PLD”,“PLD1a”, “PLD1b” or “PLD2” refer to any of the mammalian PLDs of thisinvention, “hPLD” refers to a human PLD of this invention and “mPLD”refers to a murine PLD of this invention.

[0007] Additionally, analogs of the PLD proteins and polypeptides of theinvention are provided and include truncated polypeptides, e.g., mutantsin which there are variations in the amino acid sequence that retainbiological activity, as defined below, and preferably have a homology ofat least 80%, more preferably 90%, and most preferably 95%, with thecorresponding regions of the PLD1a, PLD1b or PLD2 amino acid sequences(SEQ ID NOS:2, 5 and 8 respectively). Examples include polypeptides withminor amino acid variations from the native amino acid sequences of PLD,more specifically PLD1a, PLD1b or PLD 2 amino acid sequences (SEQ IDNOS:2, 5 and 8); in particular, conservative amino acid replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cystine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid willnot have a major effect on activity or functionality.

[0008] Using the PLD amino acid sequences of the invention (SEQ IDNOS:2, 5 and 8) other polypeptides or other DNA sequences encoding PLDproteins can be obtained. For example, the structural gene can bemanipulated by varying individual nucleotides, while retaining thecorrect amino acid(s), or varying the nucleotides, so as to modify theamino acids, without loss of activity. Nucleotides can be substituted,inserted, or deleted by known techniques, including, for example, invitro mutagenesis and primer repair. The structural gene can betruncated at its 3′-terminus and/or its 5′-terminus while retaining itsactivity. It also may be desirable to remove the region encoding thesignal sequence, and/or to replace it with a heterologous sequence. Itmay also be desirable to ligate a portion of the PLD amino acidsequences (SEQ ID NOS:2, 5 and 8), particularly that which includes theamino terminal domain to a heterologous coding sequence, and thus tocreate a fusion peptide of PLD.

[0009] In designing such modifications, it is expected that changes tononconserved regions of the PLD amino acid sequences (SEQ ID NOS:2, 5and 8) will have relatively smaller effects on activity, whereas changesin the conserved regions, and particularly in or near the amino terminaldomain are expected to produce larger effects. A residue which showsconservative variations among the PLD sequences and at least three ofthe other sequences is expected to be capable of similar conservativesubstitution of the PLD sequences. Similarly, a residue which variesnonconservatively among the PLD sequences and at least three of theother sequences is expected to be capable of either conservative ornonconservative substitution. When designing substitutions to the PLDsequences, replacement by an amino acid which is found in the comparablealigned position of one of the other sequences is especially preferred.

[0010] In another aspect, the invention provides compositions comprisinga PLD1 or PLD2 of polypeptide in combination with at least oneG-protein, for example ADP-ribosylation factor 1, RhoA, Rac1 or cdc42.

[0011] In another aspect, the invention provides novel, isolated, PLDDNA sequences not heretofore recognized or known in the art. The novelPLD DNA sequences encoding PLD1a and PLD1b proteins (SEQ ID NOS: 1 and4) were isolated from a HeLa cell line and the novel PLD DNA sequenceencoding mPLD2 protein (SEQ ID NO: 7) was isolated from a mouseembryonic cDNA library. As used herein, “isolated” means substantiallyfree from other DNA sequences with which the subject DNA is typicallyfound in its native, i.e., endogenous, state. These novel DNA sequencesare characterized by comprising the same or substantially the samenucleotide sequence as in SEQ ID NOS:1, 3, 4, 5, 7 or 9, or activefragments thereof. The DNA sequences may include 5′ and 3′ non-codingsequences flanking the coding sequence. The 5′ and 3′ non-codingsequences for hPLD1a, hPLD1b and mPLD2 are illustrated in SEQ ID NOS: 3,6 and 7 respectively. The nucleotide coding sequences only areillustrated in SEQ ID NOS: 1, 4 and 7 repsectively. The DNA sequences ofthe invention also comprise nucleotide sequences capable of hybridizingunder stringent conditions, or which would be capable of hybridizingunder said conditions but for the degeneracy of the genetic code to asequence corresponding to the sequence of SEQ ID NOS:1, 3, 4, 5, 7 or 8.SEQ ID NO:1 illustrates the DNA coding sequence of the novel PLD1a. Theputative amino acid sequence of the PLD1a protein encoded by this PLD1anucleotide sequence is illustrated in SEQ ID NO:2 and the DNA noncodingand coding sequences and putative amino acid sequence is illustrated inSEQ ID NO:3. SEQ ID NO:4 illustrates the DNA coding sequence of thenovel PLD1b. The putative amino acid sequence of the PLD1b proteinencoded by this PLD1b nucleotide sequence is illustrated in SEQ ID NO:5and the DNA noncoding and coding sequences and putative amino acidsequence of PLD1b is illustrated in SEQ ID NO: 6. SEQ ID NO:7illustrates the DNA sequence of the novel PLD2. The putative amino acidsequence of the PLD2 protein encoded by this PLD2 nucleotide sequence isillustrated in SEQ ID NO:8 and SEQ ID NO:9 illustrates the DNA noncodingand coding sequences and the putative amino acid sequence.

[0012] It is understood that the DNA sequences of this invention mayexclude some or all of the signal and/or flanking sequences. Inaddition, the DNA sequences of the present invention may also compriseDNA capable of hybridizing under stringent conditions, or which would becapable of hybridizing under such conditions but for the degeneracy ofthe genetic code, to an isolated DNA sequence of SEQ ID NOS:1, 3, 4, 6,7 or 9. As used herein, “stringent conditions” means conditions of highstringency, for example 6×SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll,0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 μg/ml salmonsperm DNA and 15% formamide at 68 degrees C.

[0013] Accordingly, the DNA sequences of this invention may containmodifications in the non-coding sequences, signal sequences or codingsequences, based on allelic variation, species or isolate variation ordeliberate modification. Using the sequences of SEQ ID NOS:1, 3, 4, 6, 7or 9, it is within the skill in the art to obtain other modified DNAsequences: the sequences can be truncated at their 3′-termini and/ortheir 5′-termini, the gene can be manipulated by varying individualnucleotides, while retaining the original amino acid(s), or varying thenucleotides, so as to modify amino acid(s). Nucleotides can besubstituted, inserted or deleted by known techniques, including forexample, in vitro mutagenesis and primer repair. In addition, short,highly degenerate oligonucleotides derived from regions of imperfectamino acid conservation can be used to identify new members of relatedfamilies. RNA molecules, transcribed from a DNA of the invention asdescribed above, are an additional aspect of the invention.

[0014] Additionally provided by this invention is a recombinant DNAvector comprising vector DNA and a DNA sequence encoding a PLDpolypeptide. The vector provides the PLD DNA in operative associationwith a regulatory sequence capable of directing the replication andexpression of a PLD protein in a selected host cell. Host cellstransformed with such vectors for use in expressing recombinant PLDproteins are also provided by this invention. Also provided is a novelprocess for producing recombinant PLD proteins or active fragmentsthereof. In this process, a host cell line transformed with a vector asdescribed above containing a DNA sequence (SEQ ID NOS: 1, 3, 4, 6, 7 or9) encoding expression of a PLD protein in operative association with asuitable regulatory sequence capable of directing replication andcontrolling expression of a PLD protein is cultured under appropriateconditions permitting expression of the recombinant DNA. The expressedprotein is then harvested from the host cell or culture medium usingsuitable conventional means. This novel process may employ various knowncells as host cell lines for expression of the protein. Currentlypreferred cells are mammalian cell lines, yeast, insect and bacterialcells. Especially preferred are insect cells and mammalian cell lines.Currently most especially preferred are baculovirus cells.

[0015] The practice of the invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA manipulation and production, and immunology, which arewithin the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Molecular Cloning; A Laboratory Manual,Second Edition (1989); DNA Cloning, Volumes I and II (D. N. Glover, Ed.1985); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, Eds. 1984); Transcriptionand Translation (B. D. Hames and S. J. Higgins, Eds. 1984); Animal CellCulture (R. I. Freshney, Ed. 1986); Immobilized Cells and Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984);the series, Methods in Enzymology (Academic Press, Inc.); Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos, Eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology, Volumes 154 and155 (Wu and Grossman, and Wu, Eds., respectively), (Mayer and Walker,Eds.) (1987); Immunochemical Methods in Cell and Molecular Biology(Academic Press, London), Scopes, (1987); Protein Purification:Principles and Practice, Second Edition (Springer-Verlag, N.Y.); andHandbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, Eds 1986). All patents, patent applications and publicationsmentioned herein, both supra and infra, are hereby incorporated byreference.

[0016] Another aspect of this invention provides pharmaceuticalcompositions for use in therapy, diagnosis, assay of PLD proteins, or inraising antibodies to PLD, comprising effective amounts of PLD proteinsprepared according to the foregoing processes.

[0017] Yet another aspect of this invention provides a method to assessPLD modulation, useful in screening for specific PLD modulatormolecules. By “PLD modulator molecule” we mean a substance that iscapable of altering the catalytic activity or the cellular location ofPLD1a, PLD1b or PLD2 under basal conditions or in the presence ofregulatory molecules, for example, by changing the action of the PLD1,PLD1b or PLD2 enzyme or by changing the affinity of PLD1, PLD1b or PLD2for its substrate. Such modulator molecules may comprise, withoutlimitation, small molecule modulators or inhibitors of PLD catalyticactivity such as small proteins, organic molecules or inorganicmolecules. Such method comprises the steps of isolating and expressing arecombinant PLD protein of the invention (and/or their active domains)and employing such PLD protein in a solid-phase assay for PLD proteinbinding. Such solid phase assays are well know in the art. Theavailability of such assays, not heretofore available, permits thedevelopment of therapeutic modulator molecules, useful in the treatmentof autoimmune or inflammatory diseases, such as for example rheumatoidarthritis, psoriasis and ulcerative colitis, in the treatment of woundhealing and other diseases or conditions characterized by exhibition ofan inflammatory response or in the treatment of cancer and otherdiseases characterized by pathogenic mitogenicity.

[0018] Further aspects of the invention therefore are pharmaceuticalcompositions containing a therapeutically effective amount of a PLDmodulator molecule identified using the assays of the invention. SuchPLD modulator molecule compositions may be employed in wound healing andin therapies for the treatment of autoimmune diseases or inflammatorydiseases, for example rheumatoid arthritis, ulcerative colitis andpsoriasis, and in the treatment of cancer and atherosclerosis, and otherdiseases characterized by exhibition of an inflammatory response or bypathogenic mitogenicity. These PLD modulator molecules may be presentedin a pharmaceutically acceptable vehicle. These pharmaceuticalcompositions may be employed alone or in combination with other suitablepharmaceutical agents, in methods for treating the aforementioneddisease states or conditions.

[0019] Such modulator molecule containing compositions may be used toinhibit neutrophil growth and differentiation, alone or in synergy withother treatment regimens such as chemotherapy and non-steroidal orsteroidal anti-inflammatory drugs. A further aspect of the inventiontherefore is a method for treating these and/or other pathologicalstates by administering to a patient a therapeutically effective amountof a PLD modulator in a suitable pharmaceutical carrier. Thesetherapeutic methods may include administering simultaneously orsequentially with a PLD modulator an effective amount of at least oneother phospholipase, cytokine, hematopoietin, interleukin, antibody,chemotherapeutic or anti-inflammatory.

[0020] Still another aspect of the invention are antibodies directedagainst PLD1a, PLD1b and/or PLD2 or a peptide thereof. Such antibodiesmay comprise PLD modulator molecules of the invention. As part of thisaspect therefore, the invention claim cell lines capable of secretingsuch antibodies and methods for their production.

[0021] Additionally provided by this invention are compositions fordetecting PLD dysfunction in mammals. These compositions comprise probeshaving at least one single-stranded fragment of at least 10 bases inlength, more preferably 15 bases in length, of a novel PLD sequence, andfragments hybridizing to these single-stranded fragments under stringenthybridization conditions and non-cross-hybridizing with mammalian DNA.Such probe compositions may additionally comprise a label, attached tothe fragment, to provide a detectable signal, as is taught in U.S. Pat.No. 4,762,780.

[0022] Further provided by this invention are methods for detecting aPLD condition in a human or other mammalian host. Such methods comprisecombining under predetermined stringency conditions a clinical samplesuspected of containing PLD DNA with at least one single-stranded DNAfragment of the novel PLD sequences having at least 10 bases, morepreferably 15 bases, and being non-cross-hybridizing with mammalian DNA,and detecting duplex formation between the single-stranded PLD fragmentsand the sample DNA. Alternatively, PCR may be used to increase thenucleic acid copy number by amplification to facilitate theidentification of PLD in individuals. In such case, the single-strandedPLD DNA sequence fragments of the present invention can be used toconstruct PCR primers for PCR-based amplification systems for thediagnosis of PLD conditions. Such systems are well known in the art. Seefor example, U.S. Pat. No. 5,008,182 (detection of AIDS associated virusby PCR) and Hedrum, PCR Methods and Applications 2:167-71(1992)(detection of Chlamydia trachomatis by PCR and immunomagnetic recovery).

[0023] Other aspects and advantages of this invention are described inthe following detailed description.

DETAILED DESCRIPTION A. Introduction

[0024] The present invention provides biologically active mammalianphospholipases, (mammalian PLDs), in forms substantially free fromassociation with other mammalian proteins and proteinaceous materialwith which they are typically found in their native state. Theseproteins can be produced by recombinant techniques to enable largequantity production of pure, active mammalian PLDs useful fortherapeutic applications. Alternatively, these proteins may be obtainedas homogeneous proteins purified from a mammalian cell line secreting orexpressing it. Further mammalian PLDs, or active fragments thereof, maybe chemically synthesized.

B. Identification of PLD DNA Sequences, Protein Characterization

[0025] Three members of the mammalian PLD family are disclosed: PLD1awas initially identified as a by product of a screening assay that haduncovered a yeast PC-specific PLD gene. See Rose, Proc. Natl. Acad. Sci.92:12151-55 (1995). The yeast PLD gene identified a GenBankhuman-expressed sequence tag (EST) encoding a significantly similarpeptide sequence. Primers were developed and HeLa cDNA was amplified byPCR (polymerase chain reaction) using oligonucleotide primers matchingthe EST. Amplification of the EST using the primers yielded a PCRproduct which was then used as a hybridization probe to screen apublicly available HeLa cDNA library at high stringency. Analysis ofpositive clones revealed a cDNA encoding what appeared to be a novel PLDenzyme. SEQ ID NO:1 illustrates the cDNA coding sequence of this clone,called PLD1a. SEQ ID NO:3 illustrates the 5′ and 3′ noncoding regionsand the cDNA coding sequence. The nucleotide sequence (SEQ ID NO:3)comprises 3609 base pairs, including a 5′ noncoding sequence of 95 basepairs, a 3′ noncoding sequence of 292 base pairs and a coding sequenceof 3222 base pairs. The PLD1a sequence is characterized by a single longopen reading frame encoding a 1074 amino acid sequence beginning withthe initiation methionine at nucleotide position 96. SEQ ID NO:2illustrated the predicted amino acid sequence of the PLD1a polypeptide.

[0026] PLD1b was initially isolated during examination of human PLD1amRNA regulation in HL-60 cells. A reverse transcription polymerase chainreaction assay (RT-PCR) was employed using primers based on the PLD1areported sequence that would amplify a central fragment of the codingregion. See Hammond, J. Biol. Chem. 270:29640-43 (1995). In addition toa PCR product of the expected size for hPLD1a, an additional and smallerfragment was amplified as well. Both fragments were cloned andsequenced. The larger band corresponded to the expected amplificationproduct, hPLD1a (SEQ ID NO:1), and the shorter product corresponded toan altered form, hPLD1b (SEQ ID NO:4), from which 114 nucleotides (38amino acids) were missing.

[0027] Using degenerate primers corresponding to the sequences encodedby the PLD1a based central region primers to amplify PLD1 from rat PC12cells and mouse embryonic cells, analogous results were obtained,demonstrating that the splice variant PLD1b (SEQ ID NOS:4 and 6) mostlikely represents an alternative splicing event of biologicalsignificance, because it is conserved in both murine and human cells.Tissue analysis shows that the “b” form predominates in mouse embryos,brain, placenta and muscle, although the “a” form is additional presentin each case.

[0028] Human PLD1b was sequenced. SEQ ID NO:4 illustrates the cDNAcoding sequence. SEQ ID NO:5 illustrates the putative amino acidsequence (single letter code). SEQ ID NO:6 illustrates the cDNAsequence, including non-coding and coding regions, and the putativeamino acid sequence (single letter code).

[0029] The nucleotide sequence of PLD1b comprises 3495 base pairs,including a 5′ noncoding sequence of 95 base pairs. The sequence alsoshows a 3′ noncoding sequence of 292 base pairs. Thus, the nucleotidesequence contains a single long reading frame of 3108 nucleotides.

[0030] The mammalian PLD1b sequence is characterized by a single longopen reading frame predicting an unprocessed 1036 amino acid polypeptidebeginning at nucleotide position 96 of SEQ ID NO:6. PLD1 and PLD2 appearstructurally dissimilar to other proteins, except other PLD proteins,with which they share similar structural features and domains. SeeMorris, Trends in Pharmacological Science 17:182-85(1996).

[0031] Mammalian PLD2 was initially isolated from a publicly availablemouse embryonic cDNA library (Stratagene) using the full length PLD1asequence (SEQ ID NO:1) as a probe to screen the library using conditionsof low stringency as described in Maniatis, Molecular Cloning (ALaboratory Manual), Cold Spring Harbor Laboratory (1982). Murine PLD2was sequenced. SEQ ID NO:7 illustrates the cDNA coding sequence. SEQ IDNO:8 illustrates the putative amino acid sequence (single letter code).SEQ ID NO:9 illustrates the cDNA sequence, including non-coding andcoding regions, and the putative amino acid sequence (single lettercode).

[0032] The nucleotide sequence of PLD2 comprises 3490 base pairs,including a 5′ noncoding sequence of 138 base pairs. The sequence alsoshows a 3′ noncoding sequence of 556 base pairs. Thus, the nucleotidesequence contains a single long reading frame of 2796 nucleotides.

[0033] The mammalian PLD2 sequence is characterized by a single longopen reading frame predicting an unprocessed 932 amino acid polypeptidebeginning at nucleotide position 139 of SEQ ID NO:9.

[0034] The nucleotide sequences of hPLD1a (SEQ ID NO:1), hPLD1b (SEQ IDNO:4) and mPLD2 (SEQ ID NO:7) have been compared with the nucleotidesequences recorded in GenBank. Other than homology with each other andother PLD proteins, no significant similarities in nucleotide sequencewere found with the published DNA sequences of other proteins. Nosignificant homology was found between the coding sequences of hPLD1a,hPLD1b or mPLD2 (SEQ ID NOS:2, 5 AND 8) and any other published non-PLDpolypeptide sequence.

[0035] Preliminary biological characterization indicates that mammalianPLD1 (SEQ ID NOS:2 or 5) is primarily associated with Golgi and otherperinuclear membrane structures and is involved in the regulation ofintravesicular membrane trafficking. PLD1 is activated by Rac1, cdc42,RhoA, PKC and ARF1, and requires PI(4,5)P₂ for activity in vitro. LikePLD1, PLD2 requires PI(4,5)P₂ for in vitro activity, but PLD2 primarilyis associated with the plasma membrane; its overexpression results in aphenotypic change in cell morphology. Cells expressing PLD2 exhibitincreases in lamellapodia formation similar in some respects tooverexpression phenotypes generated using activated cdc42, Rac1, RhoA ormembrane-targeted Ras, suggesting that PLD2 activates similarcytoskeletal reorganization pathways either in parallel or in serieswith one or more of these other activators. In further contrast to PLD1,PLD2 does not require Rac1, cdc42, RhoA, PLC or ARF1 for activation andPLD2 is down-regulated by a specific cytosolic brain inhibitor that doesnot inhibit PLD1 or PLC.

[0036] The PLD polypeptides provided herein also include polypeptidesencoded by sequences similar to that of PLD1a, PLD1b and PLD2 (SEQ IDNOS:2, 5 and 8 respectively), but into which modifications are naturallyprovided or deliberately engineered. This invention also encompassessuch novel DNA sequences, which code for expression of PLD polypeptideshaving phosphatidyl choline-specific PLD activity. These DNA sequencesinclude sequences substantially the same as the DNA sequences (SEQ IDNO: 1, 3, 4, 6, 7 and 9) and biologically active fragments thereof, andsuch sequences that hybridize under stringent hybridization conditionsto the DNA sequences (SEQ ID NOS: 1, 3, 4, 6, 7 and 9). See Maniatis,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory(1982), pages 387-389. One example of such stringent conditions ishybridization at 4×SSC, at 65 C, followed by a washing in 0.1×SSC at 65C for one hour. Another exemplary stringent hybridization scheme uses50% foramide, 4×SSC at 42 C.

[0037] DNA sequences that code for PLD polypeptides but differ in codonsequence due to the degeneracies inherent in the genetic code are alsoencompassed by this invention. Allelic variations, i.e., naturallyoccurring interspecies base changes that may or may not result in aminoacid changes, in the PLD DNA sequences (SEQ ID NOS: 1, 3, 4, 6, 7 and 9)encoding PLD polypeptides (SEQ ID NOS: 2, 5 and 8) having phosphatidylcholine-specific PLD activity are also included in this invention.

[0038] Methods for producing a desired mature polypeptide can includethe following techniques. First, a vector coding for a PLD polypeptidecan be inserted into a host cell, and the host cell can be culturedunder suitable culture conditions permitting production of thepolypeptide.

[0039] The PLD DNA sequences or active fragments thereof can beexpressed in a mammalian, insect, or microorganism host. The PLDpolynucleotides are inserted into a suitable expression vectorcompatible with the type of host cell employed and operably linked tothe control elements within that vector. Vector construction employstechniques that are known in the art. Site-specific DNA cleavageinvolved in such construction is performed by treating the vector withsuitable restriction enzymes under conditions which generally arespecified by the manufacturer of these commercially available enzymes. Asuitable expression vector is one that is compatible with the desiredfunction (e.g. transient expression, long term expression, integration,replication, amplification) and in which the control elements arecompatible with the host cell.

[0040] In order to obtain PLD expression, recombinant host cells derivedfrom transformants are incubated under conditions which allow expressionof the PLD encoding sequence (SEQ ID NOS: 1, 3, 4, 6, 7 and 9). Theseconditions will vary, depending upon the host cell elected. However, theconditions are readily ascertainable to those of ordinary skill in theart, based upon what is known in the art. Detection of a PLD proteinexpressed in the transformed host cell can be accomplished by severalmethods. For example, detection can be by enzymatic activity (orincreased enzymatic activity or increased longevity of enzymaticactivity) using fluorogenic substrates which are comprised of a dibasiccleavage site for which an PLD protein is specific. A PLD protein canalso be detected by its immunological reactivity with anti-PLDantibodies.

C. PLD Modulator Molecules

[0041] A method is provided for identifying molecules which modulate thecatalytic activity of PLD by causing a detectable loss in that activity.The method comprises transfecting a cell line with an expression vectorcomprising nucleic acid sequences encoding a PLD sequence or activedomain or fragment thereof and expressing a PLD protein. The modulatormolecule is identified by adding an effective amount of an organiccompound to the culture medium used to propagate the cells expressingthe PLD protein or active domain or fragment thereof. An effectiveamount is a concentration sufficient to block the catalysis ofphosphatidylcholine and the formation of phosphatidic acid and choline.The loss in catalytic activity may be assayed using various techniques,using intact cells or in solid-phase assays.

[0042] For example, binding assays similar to those described for IL-7in U.S. Pat. No. 5,194,375 may be used. This type of assay would involvelabeling PLD and quantifying the amount of label bound by PLD ligand inthe presence and absence of the compound being tested. The label usedmay, for example, be a radiolabel, e.g., 125I or a fluorogenic label.

[0043] Alternatively, an immunoassay may be employed to detect PLDcatalytic activity by detecting the immunological reactivity of PLD withanti-PLD antibodies in the presence and absence of the compound beingtested. The immunoassay may, for example, involve an antibody sandwichassay or an enzyme-linked immunoassay. Such methods are well known inthe art and are described in Methods in Enzymology, Vols. 154 and 155(Wu and Grossman, and Wu, Eds., respectively), (Mayer and Walker, Eds.)(1987); Immunochemical Methods in Cell and Molecular Biology (AcademicPress, London).

[0044] One assay which could be employed is disclosed in detail Example3. In such as assay the potential modulator molecule to be tested may beadded to the initial mixture or after addition of the labeled lipidmixture.

[0045] Pharmaceutical compositions comprising the PLD modulator moleculemay be used for the treatment of autoimmune diseases such as rheumatoidarthritis, psoriasis and ulcerative colitis, inflammatory diseases,wound healing and other diseases or conditions characterized byexhibition of an inflammatory response, or in the treatment of cancerand other diseases characterized by pathogenic mitogenicity. Suchpharmaceutical compositions comprise a therapeutically effective amountof one or more of the modulators in admixture with a pharmaceuticallyacceptable carrier. Other adjuvants, for instance, MF59 (Chiron Corp.),QS-21 (Cambridge Biotech Corp.), 3-DMPL (3-Deacyl-Monophosphoryl LipidA) (RibiIimmunoChem Research, Inc.), clinical grade incomplete Freund'sadjuvant (IFA), fusogenic liposomes or water soluble polymers may alsobe used. Other exemplary pharmaceutically acceptable carriers orsolutions are aluminum hydroxide, saline and phosphate buffered saline.Such pharmaceutical compositions may also contain pharmaceuticallyacceptable carriers, diluents, fillers, salts, buffers, stabilizersand/or other materials well known in the art. The term “pharmaceuticallyacceptable” means a material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s) andthat is not toxic to the host to which it is administered. Thecharacteristics of the carrier or other material will depend on theroute of administration.

[0046] Administration can be carried out in a variety of conventionalways. The composition can be systemically administered, preferablysubcutaneously or intramuscularly, in the form of an acceptablesubcutaneous or intramuscular solution. The preparation of suchsolutions, having due regard to pH, isotonicity, stability and the likeis within the skill in the art. In the long term, however, oraladministration will be advantageous, since it is expected that theactive modulator compositions will be used over a long time period totreat chronic conditions. The dosage regimen will be determined by theattending physician considering various factors known to modify theaction of drugs such as for example, physical condition, body weight,sex, diet, severity of the condition, time of administration, activityof the modulator and other clinical factors. It is currentlycontemplated, however, that the various pharmaceutical compositionsshould contain about 10 micrograms to about 1 milligram per milliliterof modulator.

[0047] In practicing the method of treatment of this invention, atherapeutically effective amount of the pharmaceutical composition isadministered to a human patient in need of such treatment. The term“therapeutically effective amount” means the total amount of the activecomponent of the method or composition that is sufficient to show ameaningful patient benefit, i.e., healing of the condition or increasein rate of healing. A therapeutically effective dose of a modulatorcomposition of this invention is contemplated to be in the range ofabout 10 micrograms to about 1 milligram per milliliter per doseadministered. The number of doses administered may vary, depending onthe individual patient and the severity of the condition.

D. Diagnostic Assays and Use as a Marker

[0048] The novel DNA sequences of the present invention can be used indiagnostic assays to detect PLD1 and/or PLD2 activity in a sample, usingeither chemically synthesized or recombinant DNA fragments. In yetanother embodiment, fragments of the DNA sequences can also be linked tosecondary nucleic acids with sequences that either bind a solid supportor other detection probes for use in diagnostic assays. In one aspect ofthe invention, fragments of the novel DNA sequences (SEQ ID NOS:1,4 and7) comprising at least between 10 and 20 nucleotides can be used asprimers to amplify nucleic acids using PCR methods well known in the artand as probes in nucleic acid hybridization assays to detect targetgenetic material such as PLD DNA in clinical specimens (with or withoutPCR). See for example, U.S. Pat. Nos. 4,683,202; 4,683,195; 5,091,310;5,008,182 and 5,168,039. In an exemplary assay, a conserved region ofthe novel DNA sequence is selected as the sequence to be amplified anddetected in the diagnostic assay. Oligonucleotide primers at leastsubstantially complementary to (but preferably identical with) thesequence to be amplified are constructed and a sample suspected ofcontaining a PLD nucleic acid sequence to be detected is treated withprimers for each strand of PLD nucleic acid sequence to be detected,four different deoxynucleotide triphosphates and a polymerization agentunder appropriate hybridization conditions such that an extensionproduct of each primer is synthesized that is complementary to the PLDnucleic acid sequences suspected in the sample, which extension productssynthesized from one primer, when separated from its complement canserve as a template for synthesis of the extension product of the otherprimer in a polymerase chain reaction. After amplification, the productof the PCR can be detected by the addition of a labeled probe, likewiseconstructed from the novel DNA sequence, capable of hybridizing with theamplified sequence as is well known in the art. See, e.g. U.S. Pat. No.5,008,182.

[0049] In another embodiment the probes or primers can be used in amarker assay to detect defects in PLD1 and/or PLD2 function.Introduction of a restriction site into the novel DNA sequence willprovide a marker that can be used with PCR fragments to detect suchdifferences in a restriction digest. Such procedures and techniques fordetecting sequence variants, such as, point mutations with the expectedlocation or configuration of the mutation, are known in the art and havebeen applied in the detection of sickle cell anemia, hemoglobin Cdisease, diabetes and other diseases and conditions as disclosed in U.S.Pat. No. 5,137,806. These methods are readily applied by one skilled inthe art to detect and differentiate between sequence variants of PLD1and/or PLD2.

[0050] In another embodiment the novel DNA sequences can be used intheir entirety or as fragments to detect the presence of DNA sequences,related sequences, or transcription products in cells, tissues, samplesand the like using hybridization probe techniques known in the art or inconjunction with one of the methods discussed herein. When used as ahybridization probe, fragments of the novel DNA sequences of theinvention are preferably 50-200 nucleotides long, more preferably100-300 nucleotides long and most preferably greater than 300nucleotides long.

E. Vectors

[0051] The novel DNA sequences of the invention can be expressed indifferent vectors using different techniques known in the art. Thevectors can be either single stranded or double stranded and made ofeither DNA or RNA. Generally, the DNA sequence is inserted into thevector alone or linked to other PLD genomic DNA. In direct in vitroligation applications, the isolated sequence alone is used. The sequence(or a fragment thereof) in a vector is operatively linked to at least apromoter and optionally an enhancer.

F. Novel Proteins

[0052] The DNA sequences, analogs or fragments thereof can be expressedin a mammalian, insect, or microorganism host. The polynucleotide isinserted into a suitable expression vector compatible with the type ofhost cell employed and is operably linked to the control elements withinthat vector. Vector construction employs techniques which are known inthe art. Site-specific DNA cleavage involved in such construction isperformed by treating with suitable restriction enzymes under conditionswhich generally are specified by the manufacturer of these commerciallyavailable enzymes. A suitable expression vector is one that iscompatible with the desired function (e.g., transient expression, longterm expression, integration, replication, amplification) and in whichthe control elements are compatible with the host cell.

[0053] Mammalian Cell Expression

[0054] Vectors suitable for replication in mammalian cells are known inthe art. Such suitable mammalian expression vectors contain a promoterto mediate transcription of foreign DNA sequences and, optionally, anenhancer. Suitable promoters are known in the art and include viralpromoters such as those from SV40, cytomegalovirus (CMV), Rous sarcomavirus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV).

[0055] The optional presence of an enhancer, combined with the promoterdescribed above, will typically increase expression levels. An enhanceris any regulatory DNA sequence that can stimulate transcription up to1000-fold when linked to endogenous or heterologous promoters, withsynthesis beginning at the normal mRNA start site. Enhancers are alsoactive when placed upstream or downstream from the transcriptioninitiation site, in either normal or flipped orientation, or at adistance of more than 1000 nucleotides from the promoter. See Maniatis,Science 236:1237(1987), Alberts, Molecular Biology of the Cell, 2nd Ed.(1989). Enhancers derived from viruses may be particularly useful,because they typically have a broader host range. Examples include theSV40 early gene enhancer (see Dijkema, EMBO J. 4:761(1985)) and theenhancer/promoters derived from the long terminal repeat (LTR) of theRSV (see Gorman, Proc. Natl. Acad. Sci. 79: 6777(1982b)) and from humancytomegalovirus (see Boshart, Cell 41: 521(1985)). Additionally, someenhancers are regulatable and become active only in the presence of aninducer, such as a hormone or metal ion (see Sassone-Corsi and Borelli,Trends Genet. 2: 215(1986)); Maniatis, Science 236: 1237(1987)). Inaddition, the expression vector can and will typically also include atermination sequence and poly(A) addition sequences which are operablylinked to the PLD coding sequence.

[0056] Sequences that cause amplification of the gene may also bedesirably included in the expression vector or in another vector that isco-translated with the expression vector containing a PLD DNA sequence,as are sequences which encode selectable markers. Selectable markers formammalian cells are known in the art, and include for example, thymidinekinase, dihydrofolate reductase (together with methotrexate as a DHFRamplifier), aminoglycoside phosphotransferase, hygromycin Bphosphotransferase, asparagine synthetase, adenosine deaminase,metallothionien, and antibiotic resistant genes such as neomycin.

[0057] The vector that encodes a novel PLD protein or polypeptide ofthis invention can be used for transformation of a suitable mammalianhost cell. Transformation can be by any known method for introducingpolynucleotide into a host cell, including, for example packaging thepolynucleotide in a virus and transducing a host cell with the virus.The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotideinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

[0058] Mammalian cell lines available as hosts for expression are knownin the art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), and a number of other cell lines.

[0059] Insect Cell Expression

[0060] The components of an insect cell expression system include atransfer vector, usually a bacterial plasmid, which contains both afragment of the baculovirus genome, and a convenient restriction sitefor insertion of the heterologous gene or genes to be expressed; a wildtype baculovirus with a sequence homologous to the baculovirus-specificfragment in the transfer vector (this allows for the homologousrecombination of the heterologous gene in to the baculovirus genome);and appropriate insect host cells and growth media. Exemplary transfervectors for introducing foreign genes into insect cells include pAc373and pVL985. See Luckow and Summers, Virology 17: 31(1989).

[0061] The plasmid can also contains the polyhedron polyadenylationsignal and a procaryotic ampicillin-resistance (amp) gene and origin ofreplication for selection and propagation in E. coli. See Miller, Ann.Rev. Microbiol. 42: 177(1988).

[0062] Baculovirus transfer vectors usually contain a baculoviruspromoter, i.e., a DNA sequence capable of binding a baculovirus RNApolymerase and initiating the downstream (5′ to 3′) transcription of acoding sequence (e.g., structural gene) into mRNA. The promoter willhave a transcription initiation region which is usually placed proximalto the 5′ end of the coding sequence and typically includes an RNApolymerase binding site and a transcription initiation site. Abaculovirus transfer vector can also have an enhancer, which, ifpresent, is usually distal to the structural gene. Expression can beeither regulated or constitutive.

[0063] A preferred baculovirus expression system employs Sf9 cells, asdetailed in Example 3.

[0064] Yeast And Bacteria Expression

[0065] A yeast expression system can typically include one or more ofthe following: a promoter sequence, fusion partner sequence, leadersequence, transcription termination sequence. A yeast promoter, capableof binding yeast RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g. structural gene) into mRNA,will have a transcription initiation region usually placed proximal tothe 5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site (a “TATA Box”) and atranscription initiation site. The yeast promoter can also have anupstream activator sequence, usually distal to the structural gene. Theactivator sequence permits inducible expression of the desiredheterologous DNA sequence. Constitutive expression occurs in the absenceof an activator sequence. Regulated expression can be either positive ornegative, thereby either enhancing or reducing transcription.

[0066] Particularly useful yeast promoters include alcohol dehydrogenase(ADH) (EP Patent Pub. No. 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK)(EP Patent Pub. No. 329 203). The yeastPHO5 gene, encoding acid phosphatase, also provides useful promotersequences. See Myanohara, Proc. Natl. Acad. Sci. 80: 1(1983).

[0067] A PLD DNA sequence, analog or an active fragment thereof can beexpressed intracellularly in yeast. A promoter sequence can be directlylinked with the sequence or fragment, in which case the first amino acidat the N-terminus of the recombinant protein will always be amethionine, which is encoded by the ATG start codon. If desired,methionine at the N-terminus can be cleaved from the protein by in vitroincubation with cyanogen bromide.

[0068] Intracellularly expressed fusion proteins provide an alternativeto direct expression of a sequence. Typically, a DNA sequence encodingthe N-terminal portion of a stable protein, a fusion partner, is fusedto the 5′ end of heterologous DNA encoding the desired polypeptide. Uponexpression, this construct will provide a fusion of the two amino acidsequences. For example, the yeast or human superoxide dismutase (SOD)gene, can be linked at the 5′ terminus of a sequence and expressed inyeast. The DNA sequence at the junction of the two amino acid sequencesmay or may not encode a clearable site. See, e.g., EP Patent Pub. No.196 056. Alternatively, the polypeptides can also be secreted from thecell into the growth media by creating a fusion protein comprised of aleader sequence fragment that provides for secretion in yeast orbacteria of the polypeptides. Preferably, there are processing sitesencoded between the leader fragment and the sequence that can be cleavedeither in vivo or in vitro. The leader sequence fragment typicallyencodes a signal peptide comprised of hydrophobic amino acids whichdirect the secretion of the protein from the cell. DNA encoding suitablesignal sequences can be derived from genes for secreted yeast proteins,such as the yeast invertase gene (EP Patent Pub. No. 12 873) and theA-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders ofnon-yeast origin, such as an interferon leader, can be used to providefor secretion in yeast (EP Patent Pub. No. 60057). Transcriptiontermination sequences recognized by yeast are regulatory regions located3′ to the translation stop codon. Together with the promoter they flankthe desired heterologous coding sequence. These flanking sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA.

[0069] Typically, the above described components, comprising a promoter,leader (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together in plasmids capable of stablemaintenance in a host, such as yeast or bacteria. The plasmid can havetwo replication systems, so it can be maintained as a shuttle vector,for example, in yeast for expression and in a procaryotic host forcloning and amplification. Examples of such yeast-bacteria shuttlevectors include YEp24 (see Botstein, Gene 8: 17-24 (1979)), pC1/1 (seeBrake, Proc. Natl. Acad. Sci. 81: 4642-4646(1984)), and YRp17 (seeStinchcomb, J. Mol. Biol. 158: 157(1982)). In addition, the plasmid canbe either a high or low copy number plasmid. A high copy number plasmidwill generally have a copy number ranging from about 5 to about 200, andtypically about 10 to about 150. A host containing a high copy numberplasmid will preferably have at least about 10, and more preferably atleast about 20. Either a high or low copy number vector may be selected,depending upon the effect on the host of the vector and thepolypeptides. See, e.g., Brake, et al., supra.

[0070] Alternatively, the expression constructs can be integrated intothe yeast genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to a yeast chromosomethat allows the vector to integrate, and preferably contain twohomologous sequences flanking the expression construct. See Orr-Weaver,Methods In Enzymol. 101: 228-245(1983) and Rine, Proc. Natl. Acad. Sci.80: 6750(1983).

[0071] Typically, extrachromosomal and integrating expression vectorscan contain selectable markers to allow for the selection of yeaststrains that have been transformed. Selectable markers can includebiosynthetic genes that can be expressed in the yeast host, such as ADE2HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which conferresistance in yeast cells to tunicamycin and G418, respectively. Inaddition, a suitable selectable marker can also provide yeast with theability to grow in the presence of toxic compounds, such as metal. Forexample, the presence of CUP1 allows yeast to grow in the presence ofcopper ions. See Butt, Microbiol. Rev. 51:351(1987).

[0072] Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors aretypically comprised of a selectable marker that is either maintained ina replicon or developed into an integrating vector, as described above.Expression and transformation vectors, either extrachromosomal orintegrating, have been developed for transformation into many yeasts.Exemplary yeasts cell lines are Candida albicans (Kurtz, Mol. Cell.Biol. 6: 142(1986), Candida maltosa (Kunze, J. Basic Microbiol. 25:141(1985), Hansenula polymorpha (Gleeson, J. Gen. Microbiol. 132:3459(1986) and Roggenkamp, Mol. Gen. Genet. 202: 302(1986),Kluyveromyces fragilis (Das, J. Bacteriol. 158: 1165(1984),Kluyveromyces lactis (De Louvencourt, J. BacterioL 154: 737(1983) andVan den Berg, Bio/Technology 8: 135(1990), Pichia guillerimondii (Kunze,J. Basic Microbiol. 25: 141(1985), Pichia pastoris (Cregg, Mol. Cell.Biol. 5: 3376 (1985), Saccharomyces cerevisiae (Hinnen, Proc. Natl.Acad. Sci. 75: 1929(1978) and Ito, J. Bacteriol. 153: 163(1983),Schizosaccharomyces pombe (Beach and Nurse, Nature 300: 706(1981), andYarrowia lipolytica (Davidow, Curr. Genet. 10: 380471(1985) andGaillardin, Curr. Genet. 10: 49(1985).

[0073] Methods of introducing exogenous DNA into yeast hosts arewell-known in the art, and typically include either the transformationof spheroplasts or of intact yeast cells treated with alkali cations.Transformation procedures usually vary with the yeast species to betransformed. See the publications listed in the foregoing paragraph forappropriate transformation techniques.

[0074] Additionally, the gene or fragment thereof can be expressed in abacterial system. In such system, a bacterial promoter is any DNAsequence capable of binding bacterial RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence (e.g. a desiredheterologous gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter can also have a second domain called an operator,that can overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein can bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression can occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation can be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli). See Raibaud, Ann. Rev. Genet. 18: 173(1984). Regulatedexpression can therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

[0075] Sequences encoding metabolic pathway enzymes provide particularlyuseful promoter sequences. Examples include promoter sequences derivedfrom sugar metabolizing enzymes, such as galactose, lactose (lac) (seeChang, Nature 198: 1056(1977), and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) (see Goeddel, Nuc. Acids Res. 8: 4057(1981), Yelverton, Nuc. AcidsRes. 9: 731(1981), U.S. Pat. No. 4,738,921 and EP Patent Pub. Nos. 36776 and 121 775). The lactomase (bla) promoter system (see Weissmann,Interferon 3 (ed. I. Gresser), the bacteriophage lambda PL promotersystem (see Shimatake, Nature 292:128(128) and the T5 promoter system(U.S. Pat. No. 4,689,406) also provides useful promoter sequences.

[0076] In addition, synthetic promoters which do not occur in naturealso function as bacterial promoters. For example, transcriptionactivation sequences of one bacterial or bacteriophage promoter can bejoined with the operon sequences of another bacterial or bacteriophagepromoter, creating a synthetic hybrid promoter such as the tac promoter(see U.S. Pat. No. 4,551,433, Amann, Gene 25: 167(1983) and de Boer,Proc. Natl. Acad. Sci. 80: 21(1983)). A bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can be coupled witha compatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is exemplary. (see Studier, J. Mol. Biol. 189: 113(1986) andTabor, Proc. Natl. Acad. Sci. 82: 1074(1985)).

[0077] In addition to a functioning promoter sequence, an efficientribosome binding site is also useful for the expression of the DNAsequence or fragment thereof in prokaryotes. In E. coli, the ribosomebinding site is called the Shine-Dalgarno (SD) sequence and includes aninitiation codon (ATG) and a sequence 3-9 nucleotides in length located3-11 nucleotides upstream of the initiation codon (see Shine, Nature254: 34(1975). The SD sequence is thought to promote binding of mRNA tothe ribosome by the pairing of bases between the SD sequence and the 3′and of E. coli 16S rRNA (see Steitz, Biological Regulation andDevelopment: Gene Expression (ed. R. F. Goldberger)(1979)).

[0078] The novel PLD proteins of the invention can be expressedintracellularly. A promoter sequence can be directly linked with a novelPLD DNA sequence, analog or a fragment thereof, in which case the firstamino acid at the N-terminus will always be a methionine, which isencoded by the ATG start codon. If desired, methionine at the N-terminuscan be cleaved from the protein by in vitro incubation with cyanogenbromide or by either in vivo on in vitro incubation with a bacterialmethionine N-terminal peptidase. See EP Patent Pub. No. 219 237.

[0079] Fusion proteins provide an alternative to direct expression.Typically, a DNA sequence encoding the N-terminal portion of anendogenous bacterial protein, or other stable protein, is fused to the5′ end of heterologous coding sequences. Upon expression, this constructwill provide a fusion of the two amino acid sequences. For example, thebacteriophage lambda cell gene can be linked at the 5′ terminus of ansequence fragment thereof and expressed in bacteria. The resultingfusion protein preferably retains a site for a processing enzyme (factorXa) to cleave the bacteriophage protein from the sequence or fragmentthereof (see Nagai, Nature 309: 810(1984). Fusion proteins can also bemade with sequences from the lacZ gene (Jia, Gene 60: 197(1987),the trpEgene (Allen, J. Biotechnol. 5: 93(1987) and Makoff, J. Gen. Microbiol.135: 11(1989), and the Chey gene (EP Patent Pub. No. 324 647) genes. TheDNA sequence at the junction of the two amino acid sequences may or maynot encode a clearable site. Another example is a ubiquitin fusionprotein. Such a fusion protein is made with the ubiquitin region thatpreferably retains a site for a processing enzyme (e.g., ubiquitinspecific processing-protease) to cleave the ubiquitin from thepolypeptide. Through this method, mature PLD1b and/or PLD2 polypeptidescan be isolated. See Miller, Bio/Technology 7: 698(1989).

[0080] Alternatively, proteins or polypeptides can also be secreted fromthe cell by creating chimeric DNA molecules that encode a fusion proteincomprised of a signal peptide sequence fragment that provides forsecretion of the proteins or polypeptides in bacteria. (See, forexample, U.S. Pat. No. 4,336,336). The signal sequence fragmenttypically encodes a signal peptide comprised of hydrophobic amino acidswhich direct the secretion of the protein from the cell. The protein iseither secreted into the growth media (gram-positive bacteria) or intothe periplasmic space, located between the inner and outer membrane ofthe cell (gram-negative bacteria). Preferably there are processingsites, which can be cleaved either in vivo or in vitro encoded betweenthe signal peptide fragment and the protein or polypeptide.

[0081] DNA encoding suitable signal sequences can be derived from genesfor secreted bacterial proteins, such as the E. coli outer membraneprotein gene (ompA) (Masui, Experimental Manipulation of Gene Expression(1983) and Ghrayeb, EMBO J. 3:2437 (1984)) and the E. coli alkalinephosphatase signal sequence (phoA) (see Oka, Proc. Natl. Acad. Sci. 82:7212 (1985). The signal sequence of the alpha-amylase gene from variousBacillus strains can be used to secrete heterologous proteins from B.subtilis (see Palva, Proc. Natl. Acad. Sci. 79: 5582 (1982) and EPPatent Pub. No. 244 042).

[0082] Transcription termination sequences recognized by bacteria areregulatory regions located 3′ to the translation stop codon. Togetherwith the promoter they flank the coding sequence. These sequences directthe transcription of an mRNA which can be translated into the PLD1band/or PLD2 protein or polypeptide encoded by the DNA sequence.Transcription termination sequences frequently include DNA sequences ofabout 50 nucleotides capable of forming stem loop structures that aid interminating transcription. Examples include transcription terminationsequences derived from genes with strong promoters, such as the trp genein E. coli as well as other biosynthetic genes.

[0083] Typically, the promoter, signal sequence (if desired), codingsequence of interest, and transcription termination sequence aremaintained in an extrachromosomal element (e.g., a plasmid) capable ofstable maintenance in the bacterial host. The plasmid will have areplication system, thus allowing it to be maintained in the bacterialhost either for expression or for cloning and amplification. Inaddition, the plasmid can be either a high or low copy number plasmid. Ahigh copy number plasmid will generally have a copy number ranging fromabout 5 to about 200, and typically about 10 to about 150. A hostcontaining a high copy number plasmid will preferably contain at leastabout 10, and more preferably at least about 20 plasmids.

[0084] Alternatively, the expression constructs can be integrated intothe bacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. See e.g., EP Patent Pub. No. 127 328.

[0085] Typically, extrachromosomal and integrating expression constructscan contain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and can include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline (see Davies, Ann. Rev. Microbiol.32: 469 (1978). Selectable markers can also include biosynthetic genes,such as those in the histidine, tryptophan, and leucine biosyntheticpathways.

[0086] Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are typicallycomprised of a selectable marker that is either maintained in anextrachromosomal vector or an integrating vector, as described above.

[0087] Expression and transformation vectors, either extra-chromosomalor integrating, have been developed for transformation into manybacteria. Exemplary are the expression vectors disclosed in Palva, Proc.Natl. Acad. Sci. 79: 5582 (1982), EP Patent Pub. Nos. 036 259 and 063953 and PCT Patent Publication WO 84/04541 (for B. subtilis); inShimatake, Nature 292: 128 (1981), Amann, Gene 40: 183 (1985), Studier,J. Mol. Biol. 189: 113 (1986) and EP Patent Pub. Nos. 036 776, 136 829and 136 907 (for E. coli); in Powell, Appl. Environ. Microbiol. 54: 655(1988) and U.S. Pat. No. 4,745,056 (for Streptococcus).

[0088] Methods of introducing exogenous DNA into bacterial hosts arewell-known in the art, and typically include either the transformationof bacteria treated with CaCl2 or other agents, such as divalent cationsand DMSO. DNA can also be introduced into bacterial cells byelectroporation. Exemplary methodologies can be found in Masson, FEMSMicrobiol. Let. 60: 273 (1989), Palva, Proc. Natl. Acad. Sci. 79: 5582(1982), EP Patent Pub. Nos. 036 259 and 063 953 and PCT Patent Pub. WO84/04541 for Bacillus transformation. For campylobacter transformation,see e.g., Miller, Proc. Natl. Acad. Sci. 85: 856 (1988) and Wang,. J.Bacteriol. 172: 949 (1990). For E. coli, see e.g., Cohen, Proc. Natl.Acad. Sci. 69: 2110 (1973), Dower, Nuc. Acids Res. 16: 6127 (1988),Kushner, Genetic Engineering: Proceedings of the International Symposiumon Genetic Engineering (eds. H. W. Boyer and S. Nicosia), Mandel, J.Mol. Biol. 53: 159 (1970) and Taketo, Biochem. Biophys. Acta 949: 318(1988). For Lactobacillus and Pseudomonas, see e.g., Chassy, FEMSMicrobiol. Let. 44: 173 (1987) and Fiedler, Anal. Biochem. 170: 38(1988), respectively. For Streptococcus, see e.g., Augustin, FEMSMicrobiol. Let. 66: 203 (1990), Barany, J. Bacteriol. 144: 698 (1980),Harlander, Streptococcal Genetics (ed. J. Ferretti and R. CurtissIII)(1987), Perry, Infec. Immun. 32: 1295 (1981), Powell, Appl. Environ.Microbiol. 54: 655 (1988) and Somkuti, Proc. 4th Evr. Cong.Biotechnology 1: 412 (1987).

[0089] The present invention is illustrated by the following examples.

MATERIALS AND METHODS Polymerase Chain Reactions (PCR)

[0090] PCRs were carried out as follows. HeLa cells were obtained fromthe American Type Culture Collection (ATCC). The cells were grown inDMEM supplemented with fetal calf serum. Total RNA was isolated from theHeLa cells by the method of Chomczynski and Sacchi. See Chomczynski,Anal. Biochem. 162: 156-59 (1987). Poly A+ RNA was obtained by affinitychromatography on oligo dT cellulose columns (Pharmacia, Piscataway,N.J.). First strand cDNA synthesis was performed starting with 5 g ofHeLa poly A+ RNA according to the manufacturer's instructions(Pharmacia).

[0091] PCR reactions were carried out for 30 cycles beginning with a1-minute incubation at 94 C, 2 minutes at 50 C, 1.5 minutes at 72 C, anda final elongation step at 72 C for 4 minutes using the PCR primersdescribed below at a final concentration of 0.25 M and HeLa cDNA atapproximately 10 ng/ml. PCR products migrating between 200 and 400 basepairs on a 1.5% agarose gel were excised, subcloned into Bluescript (sk)and manually sequenced as described by Sanger, Proc. Natl. Acad. Sci.74: 5463-67 (1977). In some instances, annealing temperatures,extensions and the number of cycles were adjusted to optimizeamplification. Sequence analysis revealed cDNAs encoding the predictedfragments upon which the primers were designed. To obtain a full-lengthversion of this clone, a bacteriophage lambda cDNA library was screened.

Nucleotide Sequence Determination and Analysis

[0092] All nucleic acid sequences were determined by thedideoxynucleotide chain termination method (Sanger et al., 1977). Avariety of templates were prepared for sequencing; they includeddouble-stranded plasmid DNA and PCR products. Manual sequencing wasemployed. The sequence was determined for both strands. Ambiguousregions were corrected by additional sequencing after proofreading. Theprimers used for sequencing were synthesized on a Model 1000 BeckmanInstruments DNA synthesizer. The contig and analysis of the sequencewere performed using MacDNASIS (Hitachi). The homology searches wereperformed using the BLAST program through NCBI services.

EXAMPLE 1 Identification and Isolation of PLD-1a

[0093] cDNAs encoding PC-specific activity were identified during ascreen that uncovered the yeast PC-specific PLD gene SP014, as describedin Rose, Proc. Natl. Acad. Sci. 92: 12151-55 (1995). Homology analysisof the yeast PLD identified a GenBank human expressed sequence tag (EST)encoding a significantly similar peptide sequence. A hybridization probewas generated from random-primed HeLa cDNA using PCR and primersspecific to the EST and used to screen a Zap II HeLa cDNA library(Stratagene). Nine hybridizing clones were detected. Sequence analysisof the hybridizing clones (using Sequenase version 2.0, U.S. BiochemicalCorp.) revealed a large open reading frame encoding a 1074 amino acidprotein. SEQ ID NOS: 1 and 2 illustrate the cDNA coding sequence andpredicted amino acid sequence (single letter code) of the human clonesof the PLD1a polypeptide. SEQ ID NO:3 illustrates the cDNA sequenceincluding 5′ and 3′ noncoding regions along with the predicted aminoacid sequence. The predicted amino acid sequence is also published inHammond, J. Biol. Chem. 270: 29640-43 (1995). The initiator methionineconforms to the eukaryotic consensus sequence and is the first in-framemethionine in the 5′ untranslated region. An in-frame stop codon islocated at nucleotides 16-17 of the cDNA, indicating that the codingregion is full length. The entire 3′ untranslated region was probablynot obtained because a recognizable polyadenylation signal sequence isnot present at the 3′ end.

[0094] Human PLD1a is devoid of heretofore recognized domain structures,in contrast to the various isoforms of PLC which contain SH2, SH3 or PHdomains. In addition, no similarity exists between human PLD1 and PLC orphosphatidylinositol-glycan-specific PLD. See Scallon, Science252:445-448 (1991). Human PLD1a bears no similarity to proteins known tobind PIP₂ or IP₃, such as PLC-, PI₃-kinase, or the IP₃ receptor.

[0095] Data base searching using human PLD1a identified homologs innumerous widely disparate organisms, demonstrating that PLD1a is amember of a novel but highly conserved gene family. Only one of thesecDNAs was recognized to encode a PC-PLD enzyme (castor bean); theremainder constitutes ESTs or hypothetical open reading frames. Acomparison of the related sequences reveals the location of severalblocks of highly conserved amino acids, one or more of which mightconstitute critical portions of the catalytic or cofactor binding sites.Two regions in particular, amino acids 455 through 490 and 892 through926, are highly conserved in all of the PLD1-related genes and containan invariant charged motif, HXKXXXXD. Mammalian PLD is believed to beintracellular and membrane-associated, as opposed to being an integralmembrane protein. Consistent with this speculation, the human PLD1aprovided herein encodes neither a signal peptide nor a hydrophobictransmembrane sequence.

EXAMPLE 2 Identification and Isolation of the hPLD1b Splice Variant

[0096] To examine hPLD1a mRNA regulation in HL-60 cells a RT-PCR assayusing primers that would amplify a central fragment of the coding regionwas conducted. HL-60 cells, maintained in RPMI-1640 medium supplementedwith 10% fetal bovine serum, 100 units/ml penicillin and 100 g/mlstreptomycin in a humidified atmosphere containing 5% CO₂, at 37 C, weregrown in RPMI-1640 medium without fetal bovine serum for 24 hours. TotalRNA was isolated from the HL-60 cells by the acid guanidine thiocyanatemethod following the method of Chomczynski, Anal. Biochem. 162:156-62(1992). Two g of RNA was reverse transcribed by using random hexamermixed primers. The number of amplification cycles were determined toindividual primer sets in order to maintain exponential rate of productamplification. Amplified DNA fragments were subjected to electrophoresison 1.5% agarose gel and visualized by ethidium bromide staining. PrimerA (Forward): nucleotides 1475 through 1491; primer B (reverse)nucleotides 2133 through 2113 of SEQ ID NO: 1. Amplification conditionsused were 94 C for 30 seconds, 60 C for 1 minute, and 72 C for 1 minute;25-27 cycles.

[0097] A PCR product of the expected size (633 nucleotides) wasgenerated, demonstrating that hPLD1a is expressed in HL-60 cells. Inaddition, an additional smaller fragment was amplified. Both fragmentswere cloned and sequenced employing the above-described methods. Thelarger band corresponded to the expected amplification product, hPLD1a.The shorter product corresponded to an altered form from which 114nucleotides (38 amino acids) were missing. Amplification withappropriately chosen junction primers permitted amplification of eachproduct independently. The cDNA coding sequence for this splice variantis illustrated in SEQ ID NO:4. The putative amino acid sequence isillustrated in SEQ ID NO:5. The cDNA sequence, including 5′ and 3′noncoding regions, along with the putative amino acid sequence isillustrated in SEQ ID NO:6. Examination of genomic sequence at theposition of amino acid 623, the 3′ junction of the missing sequence,revealed that an acceptable splice donor site, an 11 nucleotide T/C richsequence followed by any nucleotide and AGAT, was present and that theadjacent genomic sequence does not encode the nucleotide sequence foundin either the short or the long form. Thus, the long form, hPLD1a,encodes an alternately spliced exon not present in the short form,hPLD1b, and does not represent a partially processed mRNA. Subsequentanalysis of five of the original cDNAs obtained from the HeLa cell cDNAlibrary screened in Example 1 revealed that two of the cDNAs encoded thelong form and three of the cDNAs encoded the short form.

[0098] Degenerate primers corresponding to the sequences encoded byprimers A and B were used to amplify PLD1 from rat PC12 cells. Analogousresults were obtained, demonstrating that the alternate splicing eventmost likely has biological significance, since it is conserved in bothrat and human. The short form was also detected in mouse tissue andpredominated in embryos, brain, placenta and muscle, although the longform is also present in each case.

[0099] The exon is located in a “loop” region of hPLD1a which is presentin the center of the mammalian PLD1 protein but which is absent fromplant, yeast, all lower organisms, and mammalian PLD2 (see Examplebelow). Amplification of the same region of mPLD2 was carried out; noalternative splicing was observed.

EXAMPLE 3 Expression in Mammalian Cells and in Baculovirus

[0100] To investigate their catalytic properties and activationrequirements, hPLD1a and hPLD1b were expressed in Sf9 insect cells usingbaculovirus, and in COS-7 cells.

[0101] For baculovirus expression, the hPLD1a cDNA was inserted into theunique SmaI and NotI sites of the PVL1392 transfer vector (Invitrogen,Inc.) and recombinant baculoviruses harboring the cDNA were generated,selected and propagated using standard methods as described in Lucklow,Bio/Technology 6:47-55 (1988). Monolayers of Sf9 cells (30×107 cells ina 275-cm2 flask) were infected with recombinant baculoviruses at amultiplicity of 10 and cultured at 27 C for 48 hours. Expression wasdetected as follows.

[0102] The cells were washed in ice-cold phosphate-buffered saline,scraped into ice-cold lysis buffer (25 mM Tris, pH 7.5, 2 mM EDTA, 1 mMdithiothreitol, 0.1 mM benzamididine, 0.1 mM phenylmethylsulfonylfluoride), and disrupted by sonication, and the resultant suspension wascentrifuged at 2000×g for 10 minutes at 4 C. The supernatant wascentrifuged at 100,000×g for 1 hour at 4 C. The supernatant from thissecond centrifugation was removed to give the cytosolic fraction and thepellet resuspended in lysis buffer. The resuspended pellet wascentrifuged at 2000×g for 5 minutes. The supernatant obtainedconstituted the membrane fraction.

[0103] Twenty microliter samples of supernatant were collected andsubjected to a 7.5% SDS-PAGE with a Laemmli buffer system on a 7.5% geland stained with Coomassie Blue. The presence of a prominent 120 kD bandwas revealed that was not present in mock-transfected controls,consistent with the molecular mass expected for an approximately 1074amino acid protein. The identity of the 120 kD band was confirmed usinga rabbit anti-hPLD1a antisera.

[0104] Human PLD1a was also expressed in COS-7 cells. A fragment of thehPLD1a cDNA encoding the entire open reading frame (nucleotides 93through 3603 of the cDNA) was subcloned in-frame downstream of thecytomegalovirus promoter and the Flu epitope tag in the mammalianexpression vector pCGN. COS-7 cells were transfected with 3 g of theresulting plasmid, pFlu-hPLD1, using lipofectamine (Life Technologies,Inc.). Forty-eight hours later, the cells were assayed for PLD activityin the standard headgroup assay referenced below. The time course ofcholine release varied linearly over time and in proportion to theamount of protein added.

[0105] To confirm that hPLD1a encodes a PLD activity, cation-exchangeHPLC was used to analyze the water-soluble product(s) that co-elutedwith a labeled choline standard and thin layer chromatography was usedto demonstrate concurrent production of PA. Both membrane-associated andcytosolic PLD activities have been described in mammalian tissues andcell lines and it ha been suggested that the membrane-associated andcytosolic PLD activities have distinct biochemical properties and mightderive from different gene products. Samples of the reaction productswere applied to a 1 ml HPLC column of Source 15S resin (PharmaciaBiotech Inc.) and washed with 10 ml of H₂0 and eluted with a 30 mllinear gradient of 0-1 M CH₃COONH₄ in water at a flow rate of 1 ml/min.1 ml fractions were collected and their radioactivity determined byliquid scintillation counting. Compounds were identified by reference toauthentic standards. For analysis of phospholipid products, thevescicles were of standard composition except that approximately10-20×103 dpm of [³²P]PC, [³²P]PE or [³²P]PI (specific radioactivity10,000 dpm/nmol) was substituted for the [³H]PC. Incubations wereexactly as described except that transphosphatidylation assay contained2% v/v EtOH. The assays were terminated by addition of 232 l of 1:1CHCl₂, MeOH. After mixing and centrifugation, the lower phases wereremoved, dried under vacuum, and analyzed by TLC on oxalate-impregnatedWhatman 60A silica gel plates in a solvent system of CHCl₂, MeOH:aceticacid (13:3:1, v/v). Products were visualized by autoradiography andidentified by their mobilities relative to authentic standards. A bandat 120 kD was observed in the hPLD1 lane and not observed in Sf9 cellsinfected with native baculovirus vector or PLC-expressing baculovirusvector. The identity of the 120 kD band was confirmed using rabbitanti-hPLD1 antisera.

[0106] Because of the possibility that other factors in cell extractscould affect enzymatic activity, the protein was purified to homogeneityto investigate its activities in isolation. An immunoaffinity procedureusing immobilized affinity-purified anti-peptide antibodies wasdeveloped for this purpose, as follows.

[0107] a. Preparation of Affinity-Purified Anti-PLD1 Peptide Antibodies

[0108] Two rabbits were immunized with a mixture of peptidescorresponding to amino acid residues 1-15 and 525-541 of the sequence ofhPLD1 and affinity-purified antibodies (termed Ab1 and Ab525respectively) were isolated using standard procedures. These antibodiesrecognize PLD1a and PLD1b by western blotting and can immunoprecipitatetheir antigens under denaturing and non-denaturing conditions. Thepeptide antigens were chosen to generate antibodies that can distinguishPLD1 from PLD2.

[0109] b. Preparation of Immunoaffinity Resin

[0110] 1 mg of a mixture of the two affinity-purified antibodies wasadsorbed to 0.5 ml of protein-A coupled to Sepharose-Cl⁴B in phosphatebuffered saline (PBS) for 1 hour at room temperature. The resin waswashed with 0.2 M Na⁺Borate, pH 9.0 and the antibodies covalently linkedto the immobilized protein A by reaction with 20 mM dimethylpimelimidatein 0.2 M Na⁺Borate, pH 9.0 for 30 minutes at room temperature withconstant agitation. The reaction was quenched by washing the resin in0.2 M ethanolamine, pH8, after which the resin was washed extensivelyand stored in PBS containing 0.1% NaN₃.

[0111] c. Expression

[0112] Recombinant baculoviruses for expression of PLD1b were generated,selected, purified and propagated using standard techniques. Monolayersof exponentially growing Sf9 cells (3×10 7 cells/225 cm² flask, twoflasks for each purification) were infected with the viruses at amultiplicity of 10. The infected cells were grown for 48 hours, mediaremoved and washed once with ice-cold phosphate-buffered saline. Thecells were lysed on ice by addition of 5 ml/225 cm² flask of ice-coldlysis buffer containing 150 mM NaCl, 1% Nonidet P-40, 1 mM EGTA, 0.1 mMbenzamidine, 0.1 mM PMSF, 10 g/ml pepstatin A, 10 g/ml leupeptin. After30 minutes on ice, the cells were scraped up and the suspensioncentrifuged at 50,000×g for 30 minutes at 4 C. The supernatant obtained(10 mls) was mixed with 0.5 ml of the immunoaffinity resin and kept at 4C with constant agitation for 1 hour. The resin was sedimented by gentlecentrifugation and unbound protein removed. The resin was washed threetimes with 25 volumes of lysis buffer. After the final wash, the resinwas resuspended in 5 ml of lysis buffer and placed in a 10 ml BioRaddisposable column. The resin was washed with 10 ml of 10 mM phosphatebuffer, pH6.8 containing 1%-DOG as 3×0.5 ml fractions. The eluant wascollected on ice into tubes containing 0.075 ml of 1 M phosphate buffer,pH8.0. ARF-stimulated PLD activity in these fractions was determined asdescribed below and the fractions were also analyzed by SDS-PAGE andwestern blotting using standard procedures.

[0113] Expression of both PLD1a and PLD1b is considerably better whenmonolayers, as opposed to suspension cultures, of insect cells are used.In both cases however, large quantities of insoluble proteinsaccumulate. The active fraction of these recombinantly-expressed PLDenzymes is predominantly membrane-associated. The yield is approximately10 g from two 225 cm² flasks. Identical purifications from Sf9 cellsinfected with a control baculovirus produced no detectable protein bySDS-PAGE, western blotting or activity measurement.

[0114] Although the most effective extraction of the proteins requireddetergent treatment, approximately 50% of the membrane-bound PLDactivity could be extracted with 0.5 M NaCl. However, in the absence ofdetergents, enzyme activity was less stable. The purified proteins werekept at 4 C in buffer containing 1%-DOG and were stable several days.PLD1 shows a pronounced tendency to aggregate during SDS-PAGE and thisproblem is exacerbated by boiling. Sample buffer containing 8 M urea atroom temperature was therefore used to denature the proteins forSDS-PAGE.

[0115] The PLD sequence does not contain large stretches of hydrophobicresidues indicative of regions involved in mambrane insertion. PurifiedPLD1 binds to sucrose-loaded phospholipid vesicles of variouscompositions with high affinity (Kd<1 M). PLD1 does not containpleckstrin homology domains or C2 domains, protein motifs known to beinvolved in protein phospholipid interactions.

EXAMPLE 3 Catalytic Properties and Activation Requirements of HumanPLD1a and PLD1b

[0116] To determine the activity encoded by recombinant hPLD1a, controland hPLD1 a-encoding baculovirus-infected Sf9 cells were assessed usinga standard headgroup release assay that measures the amount of tritiatedheadgroup (e.g. [³H]choline) liberated by hydrolysis of the labeledsubstrate [³H]PC. The assay procedure measures release of the cholineheadgroup from the radiolabeled PC and is based on a protocol previouslydescribed in Brown, Cell 75:1137-44 (1993). Using standard separationtechniques, cytosolic and membrane fractions were prepared fromuninfected Sf9 cells or Sf9 cells infected for 48 hours with the hPLD1expressing baculovirus vector. Sf9 cells infected with nativebaculovirus vector or PLC-expressing baculovirus vector yielded modestPLD activity levels similar to untransfected cells. In contrast, Sf9cells infected with hPLD1a-encoding baculovirus exhibited substantialcytosolic (32 fold above control) and membrane-associated (15-fold abovecontrol) PLD activity.

[0117] To assess hPLD1a's substrate specificity, the standard PLD assaywas carried out using [³²P]PE and [³²P]PI following the method asdescribe in Brown, Cell 75: 1137-44 (1993). The results revealed thehPLD1a is unable to hydrolyze PE or PI. All substances exhibiting PLDactivities described to date also function as transphophatidylases inthe presence of primary alcohols, catalyzing the transfer of thephosphatidyl group from an appropriate substrate to the alcohol and thusgenerating phosphatidyl alcohol. To determine whether hPLD1a was capableof transphosphatidylase activity, EtOH was added to the reaction mixtureand the products analyzed by thin layer chromatography. The resultsdemonstrated that hPLD1a catalyzes the formation of[³²P]phosphatidylethanol when presented with [³²P]PC. Since PC-specificPLD is the only enzyme capable of catalyzing this particular reaction,hPLD1a must be a PC-specific PLD.

EXAMPLE 4

[0118] To investigate the properties of the purified PLD1 proteins indetail, the following experiments were performed.

[0119] a. Purification of G-proteins

[0120] Human ARF1 was bacterially-expressed and purified as described inRandazzo, Methods Enzymol. 257: 128-35 (1995) with a final step ofhydroxylapatite chromatography (BioRad BioGel HTP). Human RhoA, Rac1 andcdc42 were modified to contain the sequence MEEEEYMPME at the aminoterminus, expressed in Sf9 cells using baculovirus vectors, and purifiedby affinity chromatography using an immobilized monoclonal antibody asdescribed in Heyworth, Mol. Biol. Cell 4: 1217-23 (1993). The purifiedG-proteins were concentrated to 1-10 mg/ml using an Amicon pressureconcentrator with a PM-10 membrane and stored in aliquots at −80 C. Insome cases, the G-proteins were pre-activated by EDTA-stripping andloading with GTP S.

[0121] b. Purification of Protein Kinase C−

[0122] Human PKC− was expressed in Sf9 cells using a baculovirus vectorprovided by David Burns (Parke Davis Pharmaceuticals, Inc.) and purifiedfollowing the published procedure of Kitano, Methods Enzymol. 124:349-51 (1986) with minor modifications. The purified protein wasconcentrated to approximately 0.1 mg/ml using an Amicon pressureconcentrator with a PM30 membrane and stored in aliquots at −80 C.

[0123] c. PLD Assay

[0124] The basic PLD assay was performed as described in Hammond, J.Biol. Chem. 270: 29640-43 (1995) using headgroup-labeledphosphatidyl-choline. Lipids were prepared and labeled. Labeled cholinewas obtained from American Radiolabelled Chemicals Inc. (Catalog No. ART284) and extracted with chloroform and methanol 100 ml chloroform/100 mlmethanol/90 ml water (100,000 dpm/assay; 1.6 ml). The mixture wasvortexed, spun and the bottom layer collected and added to the lipidmixture to give a final concentration of 67 mM PE (Avanti Polar Lipids,Alabaster, Alabama, catalog no 850757), 7.6 mM PIP₂ (suspended in 20 mMHepes, pH 7) and 5.4 mM PC (Avanti catalog no 850457). The labeledcholine/lipid mixture was sonicated and votexed until the lipids aredissolved. Sf9 cells were plated onto large flasks (30×106 cells/flask)and left to adhere. Once adhered, medium was removed and the cells wereinfected with virus (at 1:5 or 1:10) for 1 hour with gentle rocking.After that time, virus was removed and fresh medium added, and the cellsleft to grow for 48 hours at 27 C. Medium was removed from the flask andthe cells were washed once with PBS and then lysed in 5 ml lysis buffer(25 mM Tris, 5 mM EDTA, pH 7.5). Cells were scraped out of the flask,placed on ice, sonicated for 10 seconds and centrifuged in a [type ofmachine] centrifuge for 10 minutes at 700 rpm. Supernatant was removedand spun at 30,000 rpm for 60 minutes. Supernatant from this secondcentrifugation was then removed, and the pellet was resuspended in 0.5ml lysis buffer, votexed and allowed to settle out.

[0125] For the assay, the resulting supernatant containing PLD1 enzyme(1-10 ml) was combined with G-protein(s), 20 ml assay buffer (30 mMNa-Hepes, pH 7.5, 3 mM EGTA, 80 mM KCl, 1 mM DTT, 3.0 mM MgCl₂ and 2.0mM CaCl₂) and 10 mM Hepes to make a total volume of 50 ml. 50 ml of thelabeled lipid mixture was added, vortexed thoroughly, and incubated at37 C for 30 minutes. The reaction was stopped by adding 200 ml of 10%TCA and 100 ml of 10 mg/ml BSA and centrifugation for 5 minutes at10,000 rpm. 350 ml of the supernatant was removed and counted in 2 ml ofscintillation fluid using a LKB scintillation counter for 60 seconds.This was the basic assay used for the following experiments; anymodifications were as described below.

[0126] To investigate the dependence of enzyme activity on calcium andmagnesium, the concentrations of calcium and magnesium ions of the assaymedium were varied. For some assays the lipid component of the vesicleswas altered. Ptdins (4,5)P₂ was purified from a lipid extract of bovinebrain as described in Hammond, supra. Synthetic Ptdins (3,4,5)P₃ wasobtained from Glenn Prestwich, Dept. of Chemistry, SUNY Stony Brook.[³²P]-labeled phospholipids were isolated from extracts of U937 cellslabeled overnight with [³²P] PO₄2—as described in Hammond, supra.

[0127] d. Regulation by Polyphosphoinositides

[0128] Vesicles containing 7% acidic lipid in a background of PE/PS wereemployed in the foregoing assay. The lipids tested were PI(4,5)P₂,PI(3,4,5)P₃, PI4P and PI. Only PI(4,5)P₂ and PI(3,4,5)P₃ stimulated theactivity of PLD1. Maximal activation was observed with approximately 7%PI(4,5)P₂ or PI(3,4,5)P₃, although PI(4,5)P₂ was a more effectiveactivator. At concentrations of up to 100 M, soluble inositol 1,4,5trisphosphate neither activated PLD1 nor blocked activation by PI(4,5)P₂or PI(3,4,5)P₃.

[0129] The stimulation effect for the lipids PI(4,5)P₂ and PI(3,4,5)P₃is highly selective since a variety of othr acidic phospholipids andphosphoinositides with different positional phosphate groupsubstitutions were ineffective. It is possible that the presence of alow molar fraction of PI(4,5)P₂ alters the substrate-containingphospholipid surface in a manner that renders the PC substrate morereadily hydrolyzed by the enzyme. Not all PLD activities are stimulatedby PI(4,5)P₂ and the high degree of phospholipid headgroup selectivitycoupled with the observation that PI(4,5)P₂ and PI(3,4,5)P₃ activatepure PLD1 suggests that activation involves a direct intreractionbetween PLD1 and PI(4,5)P₂. Inspection of the primary sequence of PLD1does not reveal homologies to other proteins known to interact withinositol lipids and phosphates. Phosphatidylinositol-specificphospholipase C−1 is activated by PI(4,5)P₂, which binds to anNH2-terminal non-catalytic site (a pleckstrin homology domain) anchoringthe enzymes to the membrane allowing them to function in a scooting modeof catalysis. For this reason, binding of phosphatidylinositol-specificphospholipase C−1 to phospholipid vesicles and expression of processivecatalytic activity are both markedly enhnaced by inclusion of PIP₂ inthe vesicle surface and inhibited by soluble Ins(1,4,5)P₃ which alsobinds to the NH₂-terminal PH-domain. These preliminary studies suggestthat an analogous mechanism does not underlie the activation of PLD1 byPI(4,5)P₂; that is, Ins(1,4,5)P₃ neither activates PLD1 nor inhibitsactivation by PI(4,5)P₂. PI(4,5)P₂ does not alter the binding of PLD1 tosucrose-loaded phospholipid vesicles. Another possibility is thatbinding of PI(4,5)P₂ increases enzyme activity by some allostericmechanism. In support of a direct interaction between PLD1 andPI(4,5)P₂, raedioloabeled photoreactive benzophenone-derivatives ofPI(4,5)P₂ and PI(3,4,5)P₃ can balel crude and puriried preparations ofPLD1. Since PI(4,5)P₂ and PI(3,4,5)P₃ are approximately equipotentactivators of PLD1 in vitro, given the relative abundance of these twolipids in mammalian cells, PI(4,5)P₂ is the most likely candidate for aphysiologic PLD activator.

[0130] e. Dependence on Ca²⁺ and Mg²⁺

[0131] ARF-stimulated PLD1 activity was determined as the freeconcentrations of these ions were varied in the assay medium. PLD1activity was insensitive to changes in Ca²⁺ concentration over a siderange (<10⁻⁸ M to 10⁻² M). By contrast, ARF-stimulated PLD1 activity wasdependent on Mg²⁺ with half-maximal activity observed at approximately10⁻⁴ M.

[0132] f. Activation by ARF and Rho Family G-proteins

[0133] PLD1 was incubated with increasing concentrations of purified GTPS-activated ARF1 (ADP-ribosylation factor 1), RhoA, Rac1 and cdc42.Half-maximal activation was observed with approximately 0.2 M ARF1. Thethree Rho family G-proteins were somewhat less potent activators of PLD1with half-maximal effects observed at 1 M. ARF1 was the most effectiveactivator producing an approximately 50-fold stimulation of the enzyme.RhoA and Rac1 stimulated the enzyme approximately 10- and 13-foldrespectively and cdc42 produced an approximately 5-fold activation.Effects of ARF and Rac1 were clearly saturable and, although theconcentrations of purified RhoA and cdc42 obtained limited the finalconcentrations achieved in the assay, at the highest concentrationsused, effects of these activators also appeared to be approachingsaturation. Activation of PLD1 by these G-proteins was strictlydependent on GTP S.

[0134] g. Activation by Protein Kinase C−

[0135] Purified PKC− stimulated PLD1 in a concentration dependent andsaturable manner. Half-maximal effects of PKC− were observed withapproximately 10 nM protein and the maximal effect of this activator(25-fold stimulation) was approximately 50% that observed with amaximally-effective concentration of ARF1. Inclusion of 100 mMphorbolmyristate acetate (PMA) in the assay medium increased the potencywith which PKC− stimulated PLD1 by approximately 10-fold and the maximaleffect by approximately 1.5-fold. PKC− Activation by PKC− wasATP-independent irrespective of the inclusion of PMA in the medium. When0.1 mM ATP was included in the assays, PKC-stimulated PLD activity wassomewhat lower than observed in the absence of ATP and the effect of PKCon PLD activity appeared to be more strongly dependent on PMA.Activation of PLD1 by ARF was unaffected by the inclusion of ATP. ThePKC− used in the assays exhibited phosphatidylserine and diglyceride (orPMA) stimulated autophosphorylation and phosphorylation of histone underthe same assay conditions.

[0136] h. Synergistic Effects of G-Proteins and PKC− on PLD Activity

[0137] The ARF and Rho family G-proteins and PKC− activate PLD1independently. Interactions between these regulators as activators ofpurified PLD1 was tested in the standard PLD assay.

[0138] Maximally effective concentrations of ARF1 (4.7 M), RhoA (3.8 M),Rac1 (5.3 M), cdc42 (5.5 M) and PMA-activated PKC− (0.043 M) produced49-, 13-, 13-, 10- and 28-fold activations of PLD1 respectively. Whenthis experiment was repeated including 4.7 M GTP S-activated ARF1 ineach set of assays, the response to additional ARF1 was unchanged asexpected by substantial increases in response to the Rho proteins wereobserved. In the presence of ARF1, activation by PKC− was increased to a145-fold stimulation over basal activity. Similarly, responses to Rac1and cdc42 were both increased to 106-fold of basal. Combination of PKC−with the Rho family G-proteins also produced a substantial activation ofPLD1. When combined with RhoA, Rac1 or cdc42, PKC−-stimulated PLD1activity was increased 64-, 66- and 67-fold of basal respectively whileARF1 increased the response to PKC− to a 145-fold stimulation. Bycontrast, combinations of the three Rho proteins did not result ingreater PLD1 activity than observed with each of the proteins alone. Forexample, RhoA alone produced a 13-fold activation of PLD1 and PLDactivity was not further increased by addition of concentrations of Rac1or cdc42 that were sufficient to cause a maximal activation of PLD whenadded alone. Similar observations were made for combinations of Rac1 andcdc42. Combinations of maximally-effective concentrations of ARF1 andPKC− with each of the Rho family G-proteins produced a dramatic increasein PLD1 activity. As discussed above, combination of ARF1 and PKC−stimulate PLD1 activity to a level 145-fold over basal. In the presenceof RhoA, Rac1 and cdc42, this was increased to 280-, 265- and 254-foldof basal activity.

[0139] The simplest explanation for these findings is that the PLD1protein contains separate sites for interaction with P1(4,5)P₂,PI(3,4,5)P₃, ARF, the Pho family G-proteins and protein kinase C− andthat occupancy of these sites by their respective ligands results in aco-operative increase in catalytic efficiency of the enzyme. Comparisonof the primary sequences of plant, yeast and human PLD enzymesidentifies four regions of homology including two regions containingsequences conserved among a family of related proteins that catalyzephospholipid synthesis reactions. It has been suggested that thesesequences are important in catalysis, so it appears reasonable topostulate that other regions of the protein are involved in regulatoryinteractions with the lipid and protein factors.

[0140] i. PLD1 Functions

[0141] PLD1 may be uniquely positioned to receive and integratedifferent kinds of extracellular signals, transducing them to generatelipid-derived molecules that, in turn, mediate cell-specific responses.Given the growing evidence for involvement of ARF-activated PLD inintracellular protein trafficking, receptor-regulated secretion would bea good candidate for a PLD-1 mediated response. Another, notinconsistent, possibility is that PLD1 is present in different membranecompartments of the cell where different modes of regulation predominateand different downstream effectors are present. For example,ARF-dependent activation of PLD1 in the Golgi apparatus might generatePA for coated vesicle formation while PKC and/or Rho-dependent PLDactivation of PLD1 in the plasma membrane could result in changes incell morphology mediated by the actin cytoskeleton or lead to formationof diglyceride for PKC activation.

EXAMPLE 5 Comparison of hPLD1a and hPLD1b Functional Properties

[0142] To compare activation requirements of hPLD1a and hPLD1b, bothenzymes were incubated with various combinations of activators in thepresence of labeled PC and activity determined by measuring the amountof headgroup released in the standard headgroup release assay accordingto the methods of Brown, Cell 75, supra. The activation requirements ofboth enzymes were compared in each of the assays in Example 4 above. Nodifferences in activation requirements between the hPLD1a and hPLD1bwere found.

[0143] To analyze changes in the levels of hPLD1a and hPLD1b duringHL-60 cell differentiation induced by dcbAMP, HL-60 cells maintained inRPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mlpenicillin and 100 g/ml streptomycin in a humidified atmospherecontaining 5% CO₂, at 37 C were grown in RPMI-1640 medium without fetalbovine serum for 24 hours. Differentiation was initiated by the additionof 0.5 mM dbcAMP. Levels of PLD1a and PLD1b were quantified by RT-PCR.Total RNA was isolated from the HL-60 cells at the exponential growthstage by the acid guanidine thiocyanate method following the protocol ofChomczynski, Anal. Biochem. 162: 156-62(1992). Two g of RNA was reversetranscribed by using random hexamer mixed primers. The number ofamplification cycles were determined to individual primer sets in orderto maintain exponential rate of product amplification. Amplified DNAfragments were subjected to electrophoresis on 1.5% agarose gel andvisualized by ethidium bromide staining. The intensity of bands wasquantified by a densitometer (Atto Densitograph, Tokyo, Japan). Primer A(Forward): nucleotides 1475 through 1491; primer B (reverse) nucleotides2133 through 2113 of SEQ ID NO: 1. Amplification conditions used were 94C for 30 seconds, 60 C for 1 minute, and 72 C for 1 minute; 25-27cycles.

[0144] A 3-4 fold increase in hPLD1 was observed over the 3 daydifferentiation period. Both hPLD1a and hPLD1b increased, although PLD1aincreased more rapidly. On day 1, hPLD1a exhibited an approximately3-fold increase in activity and hPLD1b exhibited a slightly less than2-fold increase in activity. On day 2, hPLD1a exhibited an approximately4-fold increase in activity and hPLD1b exhibited an approximately 3-foldincrease in activity. On day 3, hPLD1a exhibited a slightly loweredincreased activity, slightly less than 4-fold, and hPLD1b exhibited anapproximately 3.5-fold increase in activity.

[0145] Since the magnitude of rise of hPLD1 is comparable to theincrease over time of PtdBut, the increased levels of PLD1 activity canbe most simply attributed to the increased levels of hPLD1 mRNA. This isanalogous to the regulation of PLD in yeast, where levels of bothactivity and RNA are increased during meiosis when PLD activity isrequired in order for the process of sporulation to be completed. See,Rose, Proc. Natl. Acad. Sci. 92:12151-55 (1995).

EXAMPLE 6 Identification and Isolation of mPLD2, another Member of thePLD Family of Enzymes

[0146] mPLD2 was isolated using the coding sequence of hPLD1 as a probeunder conditions of reduced stringency as described in Maniatis,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory(1982) to identify related cDNAs. A 10.5 day embryonic mouse embryo cDNAlibrary and a neonatal mouse brain cDNA library (both from Stratagene,La Jolla, Calif.) were screened and approximately twenty hybridizingcDNAs were identified. Once aligned, the cDNAs represented overlappingfragments of two unique sequences that encoded unique proteins relatedto hPLD1. One sequence, denoted mPLD1, encoded a protein having sequenceidentity of approximately 96% with hPLD1. The other sequence, denotedmPLD2, encoded a protein having sequence identity of approximately 50%with hPLD1. Approximately 10% of the mPLD2 amino terminal sequence wasobtained using the RACE technique. Expressed sequence tags encoding ahuman protein having sequence identity of approximately 95% with mPLD2were later identified in GenBank. These expressed sequence tagsrepresent approximately 40% of the hPLD2 amino acid sequence.

[0147] mPLD2 activity was assessed as described in Example 3 for hPLD1.The mPLD2 protein coding sequence was subcloned in pCGN, an expressionvector containing a CMV promoter, flu-epitope tag and an SV40polyadenylation signal sequence. See Example 3. This plasmid wastransfected into COS-7 cells and cell extracts assayed for PLD activityemploying the standard headgroup assay of Example 3. mPLD2 was detectedin quantities far above COS-7 background levels. The protein codingsequence of mPLD2 was also subcloned in a baculovirus vector followingthe protocol of Example 3. mPLD2 activity was successfully detectedusing the procedures described in Example 3.

EXAMPLE 8 RNA Regulation of hPLD2

[0148] From the data of Example 7, it was known that a human analog ofmPLD2 existed. mPLD2 primers were made from the expressed sequence tagsexisting in GenBank. To analyze changes in the levels of hPLD2 activityduring HL-60 cell differentiation induced by dcbAMP, HL-60 cellsmaintained in RPMI-1640 medium supplemented with 10% fetal bovine serum,100 units.ml penicillin and 100 g/ml streptomycin in a humidifiedatmosphere containing 5% CO₂, at 37 C were grown in RPMI-1640 mediumwithout fetal bovine serum for 24 hours. Differentiation was initiatedby the addition of 0.5 mM dbcAMP. Levels of hPLD2 were quantified laterby RT-PCR. Total RNA was isolated from the HL-60 cells at theexponential growth stage by the acid guanidine thiocyanate methodfollowing the protocol of Chomczynski, Anal. Biochem. 162:156-62(1992).Two g of RNA was reverse transcribed by using random hexamer mixedprimers. The number of amplification cycles were determined toindividual primer sets in order to maintain exponential rate of productamplification. Amplified DNA fragments were subjected to electrophoresison 1.5% agarose gel and visualized by ethidium bromide staining. Theintensity of bands was quantified by a densitometer (Atto Densitograph,Tokyo, Japan). Primer E (Forward):

[0149] 5′TCCTCCAGGCCATTCTGCACT3′. Primer F (reverse):

[0150] 5′CGTTGCTCTCAGCCATGTCTTG3′. Amplification conditions used were 94C for 30 seconds, 60 C for 1 minute, and 72 C for 1 minute; 25-27cycles.

[0151] PLD2 levels rose dramatically over the three day differentiationperiod to a level approximately 20-fold higher than in undifferentiatedHL-60 cells. On day 1, hPLD2 exhibited an approximately 8-fold increasein activity. On day 2, hPLD2 exhibited an approximately 12-fold increasein activity. On day 3, hPLD2 exhibited an approximately 20-fold increasein activity.

[0152] Since total PLD activity levels did not rise as significantly,either PLD2 activation was not fully provoked by the fMLP and PMAstimuli, or the absolute amount of PLD2 is relatively small compared toPLD1.

EXAMPLE 9 Isolation and Cloning of hPLD2

[0153] hPLD2 is isolated using the coding sequence of mPLD2 as a probeunder conditions of reduced stringency as described in Maniatis,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory(1982) to identify related cDNAs. A human cDNA library is screened andhybridizing cDNAs are identified. hPLD2 activity is assessed asdescribed in Example 3 for hPLD1. The hPLD2 protein coding sequence issubcloned in pCGN, an expression vector containing a CMV promoter,flu-epitope tag and an SV40 polyadenylation signal sequence. See Example3. This plasmid is transfected into COS-7 cells and cell extractsassayed for PLD activity employing the standard headgroup assay ofExample 3. The protein coding sequence of hPLD2 can also be subcloned ina baculovirus vector following the protocol of Example 3.

EXAMPLE 10 Discussion of Results of PLD1 and PLD2 Comparisons

[0154] PLD activation has been associated with two biological processesin vivo. First, PLD is now thought to play a role in acceleratingARF-mediated bud formation in the ER and Golgi, which is required forsecretion. Second, PA, the biologically significant product of PChydrolysis by PLD, has been shown to promote filamentous actinpolymerization and stress fiber formation. A growing body of evidencesuggests that there two processes are linked such that external signalslead to increased secretion in a polarized orientation relative to theinducing signal. These events require the participation of ARF, Rho, Racand cdc42, all of which are activators of PLD1. Finally, differentiationof HL-60 cells into neutrophils is marked by increases in secretion ofchemokines, and such secretion can be blocked by the inhibition of PLDactivity. Taken together, these findings suggest that the up regulationof PLD1 may be required in order for HL-60 cells to increase theircapability as neutrophils to release chemokines in a targeted mannerupon exogenous stimulation by other immune cells.

[0155] The role of PLD2 in this process is currently unknown. More workis required to determine whether the increase in PLD1 and PLD2 isrequired for differentiated neutrophils to manifest their wellcharacterized phenotypic behavior. These results also raise the questionof whether PLD mRNA undergoes down regulation, for example afterprolonged stimulation, which is marked by a loss in recruitable PLDactivation.

[0156] The following bacterial strains containing the DNA sequences ofthe invention were deposited with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md., USA under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for purposes of patent Procedure and the Regulationsthereunder. This assures maintenance of a viable culture for 30 yearsfor the date of deposit. The organisms will be made available by theATCC under the terms of the Budapest Treaty, and subject to an agreementbetween applicant ant the ATCC that assures unrestricted availabilityupon issuance of the pertinent U.S. patent. Availability of thedeposited strains is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws. Deposit DesignationATCC No. Deposit Date Plasmid phPLD1a 97693 Aug. 26, 1996 PlasmidphPLD1b 97684 Aug. 26, 1996 Plasmid phPLD2 97695 Aug. 26, 1996

[0157] The present investigation is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and anyconstructs, viruses or enzymes which are functionally equivalent arewithin the scope of this invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

1 8 1 3222 DNA Homo sapiens 1 atgtcactga aaaacgagcc acgggtaaatacctctgcac tgcagaaaat tgctgctgac 60 atgagtaata tcatagaaaa tctggacacgcgggaactcc actttgaggg agaggaggta 120 gactacgacg tgtctcccag cgatcccaagatacaagaag tgtatatccc tttctctgct 180 atttataaca ctcaaggatt taaggagcctaatatacaga cgtatctctc cggctgtcca 240 ataaaagcac aagttctgga agtggaacgcttcacatcta caacaagggt accaagtatt 300 aatctttaca ctattgaatt aacacatggggaatttaaat ggcaagttaa gaggaaattc 360 aagcattttc aagaatttca cagagagctgctcaagtaca aagcctttat ccgcatcccc 420 attcccacta gaagacacac gtttaggaggcaaaacgtca gagaggagcc tcgagagatg 480 cccagtttgc cccgttcatc tgaaaacatgataagagaag aacaattcct tggtagaaga 540 aaacaactgg aagattactt gacaaagatactaaaaatgc ccatgtatag aaactatcat 600 gccacaacag agtttcttga tataagccagctgtctttca tccatgattt gggaccaaag 660 ggcatagaag gtatgataat gaaaagatctggaggacaca gaataccagg cttgaattgc 720 tgtggtcagg gaagagcctg ctacagatggtcaaaaagat ggttaatagt gaaagattcc 780 tttttattgt atatgaaacc agacagcggtgccattgcct tcgtcctgct ggtagacaaa 840 gaattcaaaa ttaaggtggg gaagaaggagacagaaacga aatatggaat ccgaattgat 900 aatctttcaa ggacacttat tttaaaatgcaacagctata gacatgctcg gtggtgggga 960 ggggctatag aagaattcat ccagaaacatggcaccaact ttctcaaaga tcatcgattt 1020 gggtcatatg ctgctatcca agagaatgctttagctaaat ggtatgttaa tgccaaagga 1080 tattttgaag atgtggcaaa tgcaatggaagaggcaaatg aagagatttt tatcacagac 1140 tggtggctga gtccagaaat cttcctgaaacgcccagtgg ttgagggaaa tcgttggagg 1200 ttggactgca ttcttaaacg aaaagcacaacaaggagtga ggatcttcat aatgctctac 1260 aaagaggtgg aactcgctct tggcatcaatagtgaataca ccaagaggac tttgatgcgt 1320 ctacatccca acataaaggt gatgagacacccggatcatg tgtcatccac cgtctatttg 1380 tgggctcacc atgagaagct tgtcatcattgaccaatcgg tggcctttgt gggagggatt 1440 gacctggcct atggaaggtg ggacgacaatgagcacagac tcacagacgt gggcagtgtg 1500 aagcgggtca cttcaggacc gtctctgggttccctcccac ctgccgcaat ggagtctatg 1560 gaatccttaa gactcaaaga taaaaatgagcctgttcaaa acctacccat ccagaagagt 1620 attgatgatg tggattcaaa actgaaaggaataggaaagc caagaaagtt ctccaaattt 1680 agtctctaca agcagctcca caggcaccacctgcacgacg cagatagcat cagcagcatt 1740 gacagcacct ccagttattt taatcactatagaagtcatc acaatttaat ccatggttta 1800 aaaccccact tcaaactctt tcacccgtccagtgagtctg agcaaggact cactagacct 1860 catgctgata ccgggtccat ccgtagtttacagacaggtg tgggagagct gcatggggaa 1920 accagattct ggcatggaaa ggactactgcaatttcgtct tcaaagactg ggttcaactt 1980 gataaacctt ttgctgattt cattgacaggtactccacgc cccggatgcc ctggcatgac 2040 attgcctctg cagtccacgg gaaggcggctcgtgatgtgg cacgtcactt catccagcgc 2100 tggaacttca caaaaattat gaaatcaaaatatcggtccc tttcttatcc ttttctgctt 2160 ccaaagtctc aaacaacagc ccatgagttgagatatcaag tgcctgggtc tgtccatgct 2220 aacgtacagt tgctccgctc tgctgctgattggtctgctg gtataaagta ccatgaagag 2280 tccatccacg ccgcttacgt ccatgtgatagagaacagca ggcactatat ctatatcgaa 2340 aaccagtttt tcataagctg tgctgatgacaaagttgtgt tcaacaagat aggcgatgcc 2400 attgcccaga ggatcctgaa agctcacagggaaaaccaga aataccgggt atatgtcgtg 2460 ataccacttc tgccagggtt cgaaggagacatttcaaccg gcggaggaaa tgctctacag 2520 gcaatcatgc acttcaacta cagaaccatgtgcagaggag aaaattccat ccttggacag 2580 ttaaaagcag agcttggtaa tcagtggataaattacatat cattctgtgg tcttagaaca 2640 catgcagagc tcgaaggaaa cctagtaactgagcttatct atgtccacag caagttgtta 2700 attgctgatg ataacactgt tattattggctctgccaaca taaatgaccg cagcatgctg 2760 ggaaagcgtg acagtgaaat ggctgtcattgtgcaagata cagagactgt tccttcagta 2820 atggatggaa aagagtacca agctggccggtttgcccgag gacttcggct acagtgcttt 2880 agggttgtcc ttggctatct tgatgacccaagtgaggaca ttcaggatcc agtgagtgac 2940 aaattcttca aggaggtgtg ggtttcaacagcagctcgaa atgctacaat ttatgacaag 3000 gttttccggt gccttcccaa tgatgaagtacacaatttaa ttcagctgag agactttata 3060 aacaagcccg tattagctaa ggaagatcccattcgagctg aggaggaact gaagaagatc 3120 cgtggatttt tggtgcaatt ccccttttatttcttgtctg aagaaagcct actgccttct 3180 gttgggacca aagaggccat agtgcccatggaggtttgga ct 3222 2 1074 PRT Homo sapiens 2 Met Ser Leu Lys Asn Glu ProArg Val Asn Thr Ser Ala Leu Gln Lys 1 5 10 15 Ile Ala Ala Asp Met SerAsn Ile Ile Glu Asn Leu Asp Thr Arg Glu 20 25 30 Leu His Phe Glu Gly GluGlu Val Asp Tyr Asp Val Ser Pro Ser Asp 35 40 45 Pro Lys Ile Gln Glu ValTyr Ile Pro Phe Ser Ala Ile Tyr Asn Thr 50 55 60 Gln Gly Phe Lys Glu ProAsn Ile Gln Thr Tyr Leu Ser Gly Cys Pro 65 70 75 80 Ile Lys Ala Gln ValLeu Glu Val Glu Arg Phe Thr Ser Thr Thr Arg 85 90 95 Val Pro Ser Ile AsnLeu Tyr Thr Ile Glu Leu Thr His Gly Glu Phe 100 105 110 Lys Trp Gln ValLys Arg Lys Phe Lys His Phe Gln Glu Phe His Arg 115 120 125 Glu Leu LeuLys Tyr Lys Ala Phe Ile Arg Ile Pro Ile Pro Thr Arg 130 135 140 Arg HisThr Phe Arg Arg Gln Asn Val Arg Glu Glu Pro Arg Glu Met 145 150 155 160Pro Ser Leu Pro Arg Ser Ser Glu Asn Met Ile Arg Glu Glu Gln Phe 165 170175 Leu Gly Arg Arg Lys Gln Leu Glu Asp Tyr Leu Thr Lys Ile Leu Lys 180185 190 Met Pro Met Tyr Arg Asn Tyr His Ala Thr Thr Glu Phe Leu Asp Ile195 200 205 Ser Gln Leu Ser Phe Ile His Asp Leu Gly Pro Lys Gly Ile GluGly 210 215 220 Met Ile Met Lys Arg Ser Gly Gly His Arg Ile Pro Gly LeuAsn Cys 225 230 235 240 Cys Gly Gln Gly Arg Ala Cys Tyr Arg Trp Ser LysArg Trp Leu Ile 245 250 255 Val Lys Asp Ser Phe Leu Leu Tyr Met Lys ProAsp Ser Gly Ala Ile 260 265 270 Ala Phe Val Leu Leu Val Asp Lys Glu PheLys Ile Lys Val Gly Lys 275 280 285 Lys Glu Thr Glu Thr Lys Tyr Gly IleArg Ile Asp Asn Leu Ser Arg 290 295 300 Thr Leu Ile Leu Lys Cys Asn SerTyr Arg His Ala Arg Trp Trp Gly 305 310 315 320 Gly Ala Ile Glu Glu PheIle Gln Lys His Gly Thr Asn Phe Leu Lys 325 330 335 Asp His Arg Phe GlySer Tyr Ala Ala Ile Gln Glu Asn Ala Leu Ala 340 345 350 Lys Trp Tyr ValAsn Ala Lys Gly Tyr Phe Glu Asp Val Ala Asn Ala 355 360 365 Met Glu GluAla Asn Glu Glu Ile Phe Ile Thr Asp Trp Trp Leu Ser 370 375 380 Pro GluIle Phe Leu Lys Arg Pro Val Val Glu Gly Asn Arg Trp Arg 385 390 395 400Leu Asp Cys Ile Leu Lys Arg Lys Ala Gln Gln Gly Val Arg Ile Phe 405 410415 Ile Met Leu Tyr Lys Glu Val Glu Leu Ala Leu Gly Ile Asn Ser Glu 420425 430 Tyr Thr Lys Arg Thr Leu Met Arg Leu His Pro Asn Ile Lys Val Met435 440 445 Arg His Pro Asp His Val Ser Ser Thr Val Tyr Leu Trp Ala HisHis 450 455 460 Glu Lys Leu Val Ile Ile Asp Gln Ser Val Ala Phe Val GlyGly Ile 465 470 475 480 Asp Leu Ala Tyr Gly Arg Trp Asp Asp Asn Glu HisArg Leu Thr Asp 485 490 495 Val Gly Ser Val Lys Arg Val Thr Ser Gly ProSer Leu Gly Ser Leu 500 505 510 Pro Pro Ala Ala Met Glu Ser Met Glu SerLeu Arg Leu Lys Asp Lys 515 520 525 Asn Glu Pro Val Gln Asn Leu Pro IleGln Lys Ser Ile Asp Asp Val 530 535 540 Asp Ser Lys Leu Lys Gly Ile GlyLys Pro Arg Lys Phe Ser Lys Phe 545 550 555 560 Ser Leu Tyr Lys Gln LeuHis Arg His His Leu His Asp Ala Asp Ser 565 570 575 Ile Ser Ser Ile AspSer Thr Ser Ser Tyr Phe Asn His Tyr Arg Ser 580 585 590 His His Asn LeuIle His Gly Leu Lys Pro His Phe Lys Leu Phe His 595 600 605 Pro Ser SerGlu Ser Glu Gln Gly Leu Thr Arg Pro His Ala Asp Thr 610 615 620 Gly SerIle Arg Ser Leu Gln Thr Gly Val Gly Glu Leu His Gly Glu 625 630 635 640Thr Arg Phe Trp His Gly Lys Asp Tyr Cys Asn Phe Val Phe Lys Asp 645 650655 Trp Val Gln Leu Asp Lys Pro Phe Ala Asp Phe Ile Asp Arg Tyr Ser 660665 670 Thr Pro Arg Met Pro Trp His Asp Ile Ala Ser Ala Val His Gly Lys675 680 685 Ala Ala Arg Asp Val Ala Arg His Phe Ile Gln Arg Trp Asn PheThr 690 695 700 Lys Ile Met Lys Ser Lys Tyr Arg Ser Leu Ser Tyr Pro PheLeu Leu 705 710 715 720 Pro Lys Ser Gln Thr Thr Ala His Glu Leu Arg TyrGln Val Pro Gly 725 730 735 Ser Val His Ala Asn Val Gln Leu Leu Arg SerAla Ala Asp Trp Ser 740 745 750 Ala Gly Ile Lys Tyr His Glu Glu Ser IleHis Ala Ala Tyr Val His 755 760 765 Val Ile Glu Asn Ser Arg His Tyr IleTyr Ile Glu Asn Gln Phe Phe 770 775 780 Ile Ser Cys Ala Asp Asp Lys ValVal Phe Asn Lys Ile Gly Asp Ala 785 790 795 800 Ile Ala Gln Arg Ile LeuLys Ala His Arg Glu Asn Gln Lys Tyr Arg 805 810 815 Val Tyr Val Val IlePro Leu Leu Pro Gly Phe Glu Gly Asp Ile Ser 820 825 830 Thr Gly Gly GlyAsn Ala Leu Gln Ala Ile Met His Phe Asn Tyr Arg 835 840 845 Thr Met CysArg Gly Glu Asn Ser Ile Leu Gly Gln Leu Lys Ala Glu 850 855 860 Leu GlyAsn Gln Trp Ile Asn Tyr Ile Ser Phe Cys Gly Leu Arg Thr 865 870 875 880His Ala Glu Leu Glu Gly Asn Leu Val Thr Glu Leu Ile Tyr Val His 885 890895 Ser Lys Leu Leu Ile Ala Asp Asp Asn Thr Val Ile Ile Gly Ser Ala 900905 910 Asn Ile Asn Asp Arg Ser Met Leu Gly Lys Arg Asp Ser Glu Met Ala915 920 925 Val Ile Val Gln Asp Thr Glu Thr Val Pro Ser Val Met Asp GlyLys 930 935 940 Glu Tyr Gln Ala Gly Arg Phe Ala Arg Gly Leu Arg Leu GlnCys Phe 945 950 955 960 Arg Val Val Leu Gly Tyr Leu Asp Asp Pro Ser GluAsp Ile Gln Asp 965 970 975 Pro Val Ser Asp Lys Phe Phe Lys Glu Val TrpVal Ser Thr Ala Ala 980 985 990 Arg Asn Ala Thr Ile Tyr Asp Lys Val PheArg Cys Leu Pro Asn Asp 995 1000 1005 Glu Val His Asn Leu Ile Gln LeuArg Asp Phe Ile Asn Lys Pro 1010 1015 1020 Val Leu Ala Lys Glu Asp ProIle Arg Ala Glu Glu Glu Leu Lys 1025 1030 1035 Lys Ile Arg Gly Phe LeuVal Gln Phe Pro Phe Tyr Phe Leu Ser 1040 1045 1050 Glu Glu Ser Leu LeuPro Ser Val Gly Thr Lys Glu Ala Ile Val 1055 1060 1065 Pro Met Glu ValTrp Thr 1070 3 4588 PRT Homo sapiens 3 Ala Thr Gly Thr Cys Ala Cys ThrGly Ala Ala Ala Ala Ala Cys Gly 1 5 10 15 Ala Gly Met Ser Leu Lys AsnGlu Cys Cys Ala Cys Gly Gly Gly Thr 20 25 30 Ala Ala Ala Thr Ala Cys CysThr Cys Thr Gly Cys Ala Cys Thr Gly 35 40 45 Cys Ala Gly Ala Ala Ala AlaThr Thr Gly Cys Thr Gly Cys Thr Gly 50 55 60 Ala Cys Ala Thr Gly Ala GlyThr Pro Arg Val Asn Thr Ser Ala Leu 65 70 75 80 Gln Lys Ile Ala Ala AspMet Ser Ala Ala Thr Ala Thr Cys Ala Thr 85 90 95 Ala Gly Ala Ala Ala AlaThr Cys Thr Gly Gly Ala Cys Ala Cys Gly 100 105 110 Cys Gly Gly Gly AlaAla Cys Thr Cys Cys Ala Cys Thr Thr Thr Gly 115 120 125 Ala Gly Gly GlyAla Gly Ala Gly Asn Ile Ile Glu Asn Leu Asp Thr 130 135 140 Arg Glu LeuHis Phe Glu Gly Glu Gly Ala Gly Gly Thr Ala Gly Ala 145 150 155 160 CysThr Ala Cys Gly Ala Cys Gly Thr Gly Thr Cys Thr Cys Cys Cys 165 170 175Ala Gly Cys Gly Ala Thr Cys Cys Cys Ala Ala Gly Ala Thr Ala Cys 180 185190 Ala Ala Gly Ala Ala Gly Thr Gly Glu Val Asp Tyr Asp Val Ser Pro 195200 205 Ser Asp Pro Lys Ile Gln Glu Val Thr Ala Thr Ala Thr Cys Cys Cys210 215 220 Thr Thr Thr Cys Thr Cys Thr Gly Cys Thr Ala Thr Thr Thr AlaThr 225 230 235 240 Ala Ala Cys Ala Cys Thr Cys Ala Ala Gly Gly Ala ThrThr Thr Ala 245 250 255 Ala Gly Gly Ala Gly Cys Cys Thr Tyr Ile Pro PheSer Ala Ile Tyr 260 265 270 Asn Thr Gln Gly Phe Lys Glu Pro Ala Ala ThrAla Thr Ala Cys Ala 275 280 285 Gly Ala Cys Gly Thr Ala Thr Cys Thr CysThr Cys Cys Gly Gly Cys 290 295 300 Thr Gly Thr Cys Cys Ala Ala Thr AlaAla Ala Ala Gly Cys Ala Cys 305 310 315 320 Ala Ala Gly Thr Thr Cys ThrGly Asn Ile Gln Thr Tyr Leu Ser Gly 325 330 335 Cys Pro Ile Lys Ala GlnVal Leu Gly Ala Ala Gly Thr Gly Gly Ala 340 345 350 Ala Cys Gly Cys ThrThr Cys Ala Cys Ala Thr Cys Thr Ala Cys Ala 355 360 365 Ala Cys Ala AlaGly Gly Gly Thr Ala Cys Cys Ala Ala Gly Thr Ala 370 375 380 Thr Thr AlaAla Thr Cys Thr Thr Glu Val Glu Arg Phe Thr Ser Thr 385 390 395 400 ThrArg Val Pro Ser Ile Asn Leu Thr Ala Cys Ala Cys Thr Ala Thr 405 410 415Thr Gly Ala Ala Thr Thr Ala Ala Cys Ala Cys Ala Thr Gly Gly Gly 420 425430 Gly Ala Ala Thr Thr Thr Ala Ala Ala Thr Gly Gly Cys Ala Ala Gly 435440 445 Thr Thr Ala Ala Gly Ala Gly Gly Tyr Thr Ile Glu Leu Thr His Gly450 455 460 Glu Phe Lys Trp Gln Val Lys Arg Ala Ala Ala Thr Thr Cys AlaAla 465 470 475 480 Gly Cys Ala Thr Thr Thr Thr Cys Ala Ala Gly Ala AlaThr Thr Thr 485 490 495 Cys Ala Cys Ala Gly Ala Gly Ala Gly Cys Thr GlyCys Thr Cys Ala 500 505 510 Ala Gly Thr Ala Cys Ala Ala Ala Lys Phe LysHis Phe Gln Glu Phe 515 520 525 His Arg Glu Leu Leu Lys Tyr Lys Gly CysCys Thr Thr Thr Ala Thr 530 535 540 Cys Cys Gly Cys Ala Thr Cys Cys CysCys Ala Thr Thr Cys Cys Cys 545 550 555 560 Ala Cys Thr Ala Gly Ala AlaGly Ala Cys Ala Cys Ala Cys Gly Thr 565 570 575 Thr Thr Ala Gly Gly AlaGly Gly Ala Phe Ile Arg Ile Pro Ile Pro 580 585 590 Thr Arg Arg His ThrPhe Arg Arg Cys Ala Ala Ala Ala Cys Gly Thr 595 600 605 Cys Ala Gly AlaGly Ala Gly Gly Ala Gly Cys Cys Thr Cys Gly Ala 610 615 620 Gly Ala GlyAla Thr Gly Cys Cys Cys Ala Gly Thr Thr Thr Gly Cys 625 630 635 640 CysCys Cys Gly Thr Thr Cys Ala Gln Asn Val Arg Glu Glu Pro Arg 645 650 655Glu Met Pro Ser Leu Pro Arg Ser Thr Cys Thr Gly Ala Ala Ala Ala 660 665670 Cys Ala Thr Gly Ala Thr Ala Ala Gly Ala Gly Ala Ala Gly Ala Ala 675680 685 Cys Ala Ala Thr Thr Cys Cys Thr Thr Gly Gly Thr Ala Gly Ala Ala690 695 700 Gly Ala Ala Ala Ala Cys Ala Ala Ser Glu Asn Met Ile Arg GluGlu 705 710 715 720 Gln Phe Leu Gly Arg Arg Lys Gln Cys Thr Gly Gly AlaAla Gly Ala 725 730 735 Thr Thr Ala Cys Thr Thr Gly Ala Cys Ala Ala AlaGly Ala Thr Ala 740 745 750 Cys Thr Ala Ala Ala Ala Ala Thr Gly Cys CysCys Ala Thr Gly Thr 755 760 765 Ala Thr Ala Gly Ala Ala Ala Cys Leu GluAsp Tyr Leu Thr Lys Ile 770 775 780 Leu Lys Met Pro Met Tyr Arg Asn ThrAla Thr Cys Ala Thr Gly Cys 785 790 795 800 Cys Ala Cys Ala Ala Cys AlaGly Ala Gly Thr Thr Thr Cys Thr Thr 805 810 815 Gly Ala Thr Ala Thr AlaAla Gly Cys Cys Ala Gly Cys Thr Gly Thr 820 825 830 Cys Thr Thr Thr CysAla Thr Cys Tyr His Ala Thr Thr Glu Phe Leu 835 840 845 Asp Ile Ser GlnLeu Ser Phe Ile Cys Ala Thr Gly Ala Thr Thr Thr 850 855 860 Gly Gly GlyAla Cys Cys Ala Ala Ala Gly Gly Gly Cys Ala Thr Ala 865 870 875 880 GlyAla Ala Gly Gly Thr Ala Thr Gly Ala Thr Ala Ala Thr Gly Ala 885 890 895Ala Ala Ala Gly Ala Thr Cys Thr His Asp Leu Gly Pro Lys Gly Ile 900 905910 Glu Gly Met Ile Met Lys Arg Ser Gly Gly Ala Gly Gly Ala Cys Ala 915920 925 Cys Ala Gly Ala Ala Thr Ala Cys Cys Ala Gly Gly Cys Thr Thr Gly930 935 940 Ala Ala Thr Thr Gly Cys Thr Gly Thr Gly Gly Thr Cys Ala GlyGly 945 950 955 960 Gly Ala Ala Gly Ala Gly Cys Cys Gly Gly His Arg IlePro Gly Leu 965 970 975 Asn Cys Cys Gly Gln Gly Arg Ala Thr Gly Cys ThrAla Cys Ala Gly 980 985 990 Ala Thr Gly Gly Thr Cys Ala Ala Ala Ala AlaGly Ala Thr Gly Gly 995 1000 1005 Thr Thr Ala Ala Thr Ala Gly Thr GlyAla Ala Ala Gly Ala Thr 1010 1015 1020 Thr Cys Cys Thr Thr Thr Thr ThrAla Cys Tyr Arg Trp Ser Lys 1025 1030 1035 Arg Trp Leu Ile Val Lys AspSer Phe Leu Thr Thr Gly Thr Ala 1040 1045 1050 Thr Ala Thr Gly Ala AlaAla Cys Cys Ala Gly Ala Cys Ala Gly 1055 1060 1065 Cys Gly Gly Thr GlyCys Cys Ala Thr Thr Gly Cys Cys Thr Thr 1070 1075 1080 Cys Gly Thr CysCys Thr Gly Cys Thr Gly Gly Thr Ala Leu Tyr 1085 1090 1095 Met Lys ProAsp Ser Gly Ala Ile Ala Phe Val Leu Leu Val Gly 1100 1105 1110 Ala CysAla Ala Ala Gly Ala Ala Thr Thr Cys Ala Ala Ala Ala 1115 1120 1125 ThrThr Ala Ala Gly Gly Thr Gly Gly Gly Gly Ala Ala Gly Ala 1130 1135 1140Ala Gly Gly Ala Gly Ala Cys Ala Gly Ala Ala Ala Cys Gly Ala 1145 11501155 Ala Ala Asp Lys Glu Phe Lys Ile Lys Val Gly Lys Lys Glu Thr 11601165 1170 Glu Thr Lys Thr Ala Thr Gly Gly Ala Ala Thr Cys Cys Gly Ala1175 1180 1185 Ala Thr Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Thr CysAla 1190 1195 1200 Ala Gly Gly Ala Cys Ala Cys Thr Thr Ala Thr Thr ThrThr Ala 1205 1210 1215 Ala Ala Ala Thr Gly Cys Tyr Gly Ile Arg Ile AspAsn Leu Ser 1220 1225 1230 Arg Thr Leu Ile Leu Lys Cys Ala Ala Cys AlaGly Cys Thr Ala 1235 1240 1245 Thr Ala Gly Ala Cys Ala Thr Gly Cys ThrCys Gly Gly Thr Gly 1250 1255 1260 Gly Thr Gly Gly Gly Gly Ala Gly GlyGly Gly Cys Thr Ala Thr 1265 1270 1275 Ala Gly Ala Ala Gly Ala Ala ThrThr Cys Asn Ser Tyr Arg His 1280 1285 1290 Ala Arg Trp Trp Gly Gly AlaIle Glu Glu Phe Ala Thr Cys Cys 1295 1300 1305 Ala Gly Ala Ala Ala CysAla Thr Gly Gly Cys Ala Cys Cys Ala 1310 1315 1320 Ala Cys Thr Thr ThrCys Thr Cys Ala Ala Ala Gly Ala Thr Cys 1325 1330 1335 Ala Thr Cys GlyAla Thr Thr Thr Gly Gly Gly Thr Cys Ala Ile 1340 1345 1350 Gln Lys HisGly Thr Asn Phe Leu Lys Asp His Arg Phe Gly Ser 1355 1360 1365 Thr AlaThr Gly Cys Thr Gly Cys Thr Ala Thr Cys Cys Ala Ala 1370 1375 1380 GlyAla Gly Ala Ala Thr Gly Cys Thr Thr Thr Ala Gly Cys Thr 1385 1390 1395Ala Ala Ala Thr Gly Gly Thr Ala Thr Gly Thr Thr Ala Ala Thr 1400 14051410 Gly Cys Cys Tyr Ala Ala Ile Gln Glu Asn Ala Leu Ala Lys Trp 14151420 1425 Tyr Val Asn Ala Ala Ala Ala Gly Gly Ala Thr Ala Thr Thr Thr1430 1435 1440 Thr Gly Ala Ala Gly Ala Thr Gly Thr Gly Gly Cys Ala AlaAla 1445 1450 1455 Thr Gly Cys Ala Ala Thr Gly Gly Ala Ala Gly Ala GlyGly Cys 1460 1465 1470 Ala Ala Ala Thr Gly Ala Ala Lys Gly Tyr Phe GluAsp Val Ala 1475 1480 1485 Asn Ala Met Glu Glu Ala Asn Glu Gly Ala GlyAla Thr Thr Thr 1490 1495 1500 Thr Thr Ala Thr Cys Ala Cys Ala Gly AlaCys Thr Gly Gly Thr 1505 1510 1515 Gly Gly Cys Thr Gly Ala Gly Thr CysCys Ala Gly Ala Ala Ala 1520 1525 1530 Thr Cys Thr Thr Cys Cys Thr GlyAla Ala Ala Glu Ile Phe Ile 1535 1540 1545 Thr Asp Trp Trp Leu Ser ProGlu Ile Phe Leu Lys Cys Gly Cys 1550 1555 1560 Cys Cys Ala Gly Thr GlyGly Thr Thr Gly Ala Gly Gly Gly Ala 1565 1570 1575 Ala Ala Thr Cys GlyThr Thr Gly Gly Ala Gly Gly Thr Thr Gly 1580 1585 1590 Gly Ala Cys ThrGly Cys Ala Thr Thr Cys Thr Thr Ala Ala Ala 1595 1600 1605 Arg Pro ValVal Glu Gly Asn Arg Trp Arg Leu Asp Cys Ile Leu 1610 1615 1620 Lys CysGly Ala Ala Ala Ala Gly Cys Ala Cys Ala Ala Cys Ala 1625 1630 1635 AlaGly Gly Ala Gly Thr Gly Ala Gly Gly Ala Thr Cys Thr Thr 1640 1645 1650Cys Ala Thr Ala Ala Thr Gly Cys Thr Cys Thr Ala Cys Ala Ala 1655 16601665 Ala Gly Ala Gly Arg Lys Ala Gln Gln Gly Val Arg Ile Phe Ile 16701675 1680 Met Leu Tyr Lys Glu Gly Thr Gly Gly Ala Ala Cys Thr Cys Gly1685 1690 1695 Cys Thr Cys Thr Thr Gly Gly Cys Ala Thr Cys Ala Ala ThrAla 1700 1705 1710 Gly Thr Gly Ala Ala Thr Ala Cys Ala Cys Cys Ala AlaGly Ala 1715 1720 1725 Gly Gly Ala Cys Thr Thr Thr Gly Val Glu Leu AlaLeu Gly Ile 1730 1735 1740 Asn Ser Glu Tyr Thr Lys Arg Thr Leu Ala ThrGly Cys Gly Thr 1745 1750 1755 Cys Thr Ala Cys Ala Thr Cys Cys Cys AlaAla Cys Ala Thr Ala 1760 1765 1770 Ala Ala Gly Gly Thr Gly Ala Thr GlyAla Gly Ala Cys Ala Cys 1775 1780 1785 Cys Cys Gly Gly Ala Thr Cys AlaThr Gly Thr Gly Met Arg Leu 1790 1795 1800 His Pro Asn Ile Lys Val MetArg His Pro Asp His Val Thr Cys 1805 1810 1815 Ala Thr Cys Cys Ala CysCys Gly Thr Cys Thr Ala Thr Thr Thr 1820 1825 1830 Gly Thr Gly Gly GlyCys Thr Cys Ala Cys Cys Ala Thr Gly Ala 1835 1840 1845 Gly Ala Ala GlyCys Thr Thr Gly Thr Cys Ala Thr Cys Ala Thr 1850 1855 1860 Thr Ser SerThr Val Tyr Leu Trp Ala His His Glu Lys Leu Val 1865 1870 1875 Ile IleGly Ala Cys Cys Ala Ala Thr Cys Gly Gly Thr Gly Gly 1880 1885 1890 CysCys Thr Thr Thr Gly Thr Gly Gly Gly Ala Gly Gly Gly Ala 1895 1900 1905Thr Thr Gly Ala Cys Cys Thr Gly Gly Cys Cys Thr Ala Thr Gly 1910 19151920 Gly Ala Ala Gly Gly Asp Gln Ser Val Ala Phe Val Gly Gly Ile 19251930 1935 Asp Leu Ala Tyr Gly Arg Thr Gly Gly Gly Ala Cys Gly Ala Cys1940 1945 1950 Ala Ala Thr Gly Ala Gly Cys Ala Cys Ala Gly Ala Cys ThrCys 1955 1960 1965 Ala Cys Ala Gly Ala Cys Gly Thr Gly Gly Gly Cys AlaGly Thr 1970 1975 1980 Gly Thr Gly Ala Ala Gly Cys Gly Gly Trp Asp AspAsn Glu His 1985 1990 1995 Arg Leu Thr Asp Val Gly Ser Val Lys Arg GlyThr Cys Ala Cys 2000 2005 2010 Thr Thr Cys Ala Gly Gly Ala Cys Cys GlyThr Cys Thr Cys Thr 2015 2020 2025 Gly Gly Gly Thr Thr Cys Cys Cys ThrCys Cys Cys Ala Cys Cys 2030 2035 2040 Thr Gly Cys Cys Gly Cys Ala AlaThr Gly Gly Ala Gly Val Thr 2045 2050 2055 Ser Gly Pro Ser Leu Gly SerLeu Pro Pro Ala Ala Met Glu Thr 2060 2065 2070 Cys Thr Ala Thr Gly GlyAla Ala Thr Cys Cys Thr Thr Ala Ala 2075 2080 2085 Gly Ala Cys Thr CysAla Ala Ala Gly Ala Thr Ala Ala Ala Ala 2090 2095 2100 Ala Thr Gly AlaGly Cys Cys Thr Gly Thr Thr Cys Ala Ala Ala 2105 2110 2115 Ala Cys SerMet Glu Ser Leu Arg Leu Lys Asp Lys Asn Glu Pro 2120 2125 2130 Val GlnAsn Cys Thr Ala Cys Cys Cys Ala Thr Cys Cys Ala Gly 2135 2140 2145 AlaAla Gly Ala Gly Thr Ala Thr Thr Gly Ala Thr Gly Ala Thr 2150 2155 2160Gly Thr Gly Gly Ala Thr Thr Cys Ala Ala Ala Ala Cys Thr Gly 2165 21702175 Ala Ala Ala Gly Gly Ala Leu Pro Ile Gln Lys Ser Ile Asp Asp 21802185 2190 Val Asp Ser Lys Leu Lys Gly Ala Thr Ala Gly Gly Ala Ala Ala2195 2200 2205 Gly Cys Cys Ala Ala Gly Ala Ala Ala Gly Thr Thr Cys ThrCys 2210 2215 2220 Cys Ala Ala Ala Thr Thr Thr Ala Gly Thr Cys Thr CysThr Ala 2225 2230 2235 Cys Ala Ala Gly Cys Ala Gly Cys Thr Cys Ile GlyLys Pro Arg 2240 2245 2250 Lys Phe Ser Lys Phe Ser Leu Tyr Lys Gln LeuCys Ala Cys Ala 2255 2260 2265 Gly Gly Cys Ala Cys Cys Ala Cys Cys ThrGly Cys Ala Cys Gly 2270 2275 2280 Ala Cys Gly Cys Ala Gly Ala Thr AlaGly Cys Ala Thr Cys Ala 2285 2290 2295 Gly Cys Ala Gly Cys Ala Thr ThrGly Ala Cys Ala Gly Cys His 2300 2305 2310 Arg His His Leu His Asp AlaAsp Ser Ile Ser Ser Ile Asp Ser 2315 2320 2325 Ala Cys Cys Thr Cys CysAla Gly Thr Thr Ala Thr Thr Thr Thr 2330 2335 2340 Ala Ala Thr Cys AlaCys Thr Ala Thr Ala Gly Ala Ala Gly Thr 2345 2350 2355 Cys Ala Thr CysAla Cys Ala Ala Thr Thr Thr Ala Ala Thr Cys 2360 2365 2370 Cys Ala ThrThr Ser Ser Tyr Phe Asn His Tyr Arg Ser His His 2375 2380 2385 Asn LeuIle His Gly Gly Thr Thr Thr Ala Ala Ala Ala Cys Cys 2390 2395 2400 CysCys Ala Cys Thr Thr Cys Ala Ala Ala Cys Thr Cys Thr Thr 2405 2410 2415Thr Cys Ala Cys Cys Cys Gly Thr Cys Cys Ala Gly Thr Gly Ala 2420 24252430 Gly Thr Cys Thr Gly Ala Gly Gly Leu Lys Pro His Phe Lys Leu 24352440 2445 Phe His Pro Ser Ser Glu Ser Glu Cys Ala Ala Gly Gly Ala Cys2450 2455 2460 Thr Cys Ala Cys Thr Ala Gly Ala Cys Cys Thr Cys Ala ThrGly 2465 2470 2475 Cys Thr Gly Ala Thr Ala Cys Cys Gly Gly Gly Thr CysCys Ala 2480 2485 2490 Thr Cys Cys Gly Thr Ala Gly Thr Thr Thr Ala GlnGly Leu Thr 2495 2500 2505 Arg Pro His Ala Asp Thr Gly Ser Ile Arg SerLeu Cys Ala Gly 2510 2515 2520 Ala Cys Ala Gly Gly Thr Gly Thr Gly GlyGly Ala Gly Ala Gly 2525 2530 2535 Cys Thr Gly Cys Ala Thr Gly Gly GlyGly Ala Ala Ala Cys Cys 2540 2545 2550 Ala Gly Ala Thr Thr Cys Thr GlyGly Cys Ala Thr Gly Gly Ala 2555 2560 2565 Gln Thr Gly Val Gly Glu LeuHis Gly Glu Thr Arg Phe Trp His 2570 2575 2580 Gly Ala Ala Gly Gly AlaCys Thr Ala Cys Thr Gly Cys Ala Ala 2585 2590 2595 Thr Thr Thr Cys GlyThr Cys Thr Thr Cys Ala Ala Ala Gly Ala 2600 2605 2610 Cys Thr Gly GlyGly Thr Thr Cys Ala Ala Cys Thr Thr Gly Ala 2615 2620 2625 Thr Ala AlaAla Lys Asp Tyr Cys Asn Phe Val Phe Lys Asp Trp 2630 2635 2640 Val GlnLeu Asp Lys Cys Cys Thr Thr Thr Thr Gly Cys Thr Gly 2645 2650 2655 AlaThr Thr Thr Cys Ala Thr Thr Gly Ala Cys Ala Gly Gly Thr 2660 2665 2670Ala Cys Thr Cys Cys Ala Cys Gly Cys Cys Cys Cys Gly Gly Ala 2675 26802685 Thr Gly Cys Cys Cys Thr Gly Gly Pro Phe Ala Asp Phe Ile Asp 26902695 2700 Arg Tyr Ser Thr Pro Arg Met Pro Trp Cys Ala Thr Gly Ala Cys2705 2710 2715 Ala Thr Thr Gly Cys Cys Thr Cys Thr Gly Cys Ala Gly ThrCys 2720 2725 2730 Cys Ala Cys Gly Gly Gly Ala Ala Gly Gly Cys Gly GlyCys Thr 2735 2740 2745 Cys Gly Thr Gly Ala Thr Gly Thr Gly Gly Cys AlaHis Asp Ile 2750 2755 2760 Ala Ser Ala Val His Gly Lys Ala Ala Arg AspVal Ala Cys Gly 2765 2770 2775 Thr Cys Ala Cys Thr Thr Cys Ala Thr CysCys Ala Gly Cys Gly 2780 2785 2790 Cys Thr Gly Gly Ala Ala Cys Thr ThrCys Ala Cys Ala Ala Ala 2795 2800 2805 Ala Ala Thr Thr Ala Thr Gly AlaAla Ala Thr Cys Ala Ala Ala 2810 2815 2820 Ala Arg His Phe Ile Gln ArgTrp Asn Phe Thr Lys Ile Met Lys 2825 2830 2835 Ser Lys Thr Ala Thr CysGly Gly Thr Cys Cys Cys Thr Thr Thr 2840 2845 2850 Cys Thr Thr Ala ThrCys Cys Thr Thr Thr Thr Cys Thr Gly Cys 2855 2860 2865 Thr Thr Cys CysAla Ala Ala Gly Thr Cys Thr Cys Ala Ala Ala 2870 2875 2880 Cys Ala AlaCys Ala Tyr Arg Ser Leu Ser Tyr Pro Phe Leu Leu 2885 2890 2895 Pro LysSer Gln Thr Thr Gly Cys Cys Cys Ala Thr Gly Ala Gly 2900 2905 2910 ThrThr Gly Ala Gly Ala Thr Ala Thr Cys Ala Ala Gly Thr Gly 2915 2920 2925Cys Cys Thr Gly Gly Gly Thr Cys Thr Gly Thr Cys Cys Ala Thr 2930 29352940 Gly Cys Thr Ala Ala Cys Gly Thr Ala Ala His Glu Leu Arg Tyr 29452950 2955 Gln Val Pro Gly Ser Val His Ala Asn Val Cys Ala Gly Thr Thr2960 2965 2970 Gly Cys Thr Cys Cys Gly Cys Thr Cys Thr Gly Cys Thr GlyCys 2975 2980 2985 Thr Gly Ala Thr Thr Gly Gly Thr Cys Thr Gly Cys ThrGly Gly 2990 2995 3000 Thr Ala Thr Ala Ala Ala Gly Thr Ala Cys Cys AlaThr Gln Leu 3005 3010 3015 Leu Arg Ser Ala Ala Asp Trp Ser Ala Gly IleLys Tyr His Gly 3020 3025 3030 Ala Ala Gly Ala Gly Thr Cys Cys Ala ThrCys Cys Ala Cys Gly 3035 3040 3045 Cys Cys Gly Cys Thr Thr Ala Cys GlyThr Cys Cys Ala Thr Gly 3050 3055 3060 Thr Gly Ala Thr Ala Gly Ala GlyAla Ala Cys Ala Gly Cys Ala 3065 3070 3075 Gly Gly Glu Glu Ser Ile HisAla Ala Tyr Val His Val Ile Glu 3080 3085 3090 Asn Ser Arg Cys Ala CysThr Ala Thr Ala Thr Cys Thr Ala Thr 3095 3100 3105 Ala Thr Cys Gly AlaAla Ala Ala Cys Cys Ala Gly Thr Thr Thr 3110 3115 3120 Thr Thr Cys AlaThr Ala Ala Gly Cys Thr Gly Thr Gly Cys Thr 3125 3130 3135 Gly Ala ThrGly Ala Cys His Tyr Ile Tyr Ile Glu Asn Gln Phe 3140 3145 3150 Phe IleSer Cys Ala Asp Asp Ala Ala Ala Gly Thr Thr Gly Thr 3155 3160 3165 GlyThr Thr Cys Ala Ala Cys Ala Ala Gly Ala Thr Ala Gly Gly 3170 3175 3180Cys Gly Ala Thr Gly Cys Cys Ala Thr Thr Gly Cys Cys Cys Ala 3185 31903195 Gly Ala Gly Gly Ala Thr Cys Cys Thr Gly Lys Val Val Phe Asn 32003205 3210 Lys Ile Gly Asp Ala Ile Ala Gln Arg Ile Leu Ala Ala Ala Gly3215 3220 3225 Cys Thr Cys Ala Cys Ala Gly Gly Gly Ala Ala Ala Ala CysCys 3230 3235 3240 Ala Gly Ala Ala Ala Thr Ala Cys Cys Gly Gly Gly ThrAla Thr 3245 3250 3255 Ala Thr Gly Thr Cys Gly Thr Gly Ala Thr Ala CysCys Ala Lys 3260 3265 3270 Ala His Arg Glu Asn Gln Lys Tyr Arg Val TyrVal Val Ile Pro 3275 3280 3285 Cys Thr Thr Cys Thr Gly Cys Cys Ala GlyGly Gly Thr Thr Cys 3290 3295 3300 Gly Ala Ala Gly Gly Ala Gly Ala CysAla Thr Thr Thr Cys Ala 3305 3310 3315 Ala Cys Cys Gly Gly Cys Gly GlyAla Gly Gly Ala Ala Ala Thr 3320 3325 3330 Gly Cys Thr Leu Leu Pro GlyPhe Glu Gly Asp Ile Ser Thr Gly 3335 3340 3345 Gly Gly Asn Ala Cys ThrAla Cys Ala Gly Gly Cys Ala Ala Thr 3350 3355 3360 Cys Ala Thr Gly CysAla Cys Thr Thr Cys Ala Ala Cys Thr Ala 3365 3370 3375 Cys Ala Gly AlaAla Cys Cys Ala Thr Gly Thr Gly Cys Ala Gly 3380 3385 3390 Ala Gly GlyAla Gly Ala Ala Leu Gln Ala Ile Met His Phe Asn 3395 3400 3405 Tyr ArgThr Met Cys Arg Gly Glu Ala Ala Thr Thr Cys Cys Ala 3410 3415 3420 ThrCys Cys Thr Thr Gly Gly Ala Cys Ala Gly Thr Thr Ala Ala 3425 3430 3435Ala Ala Gly Cys Ala Gly Ala Gly Cys Thr Thr Gly Gly Thr Ala 3440 34453450 Ala Thr Cys Ala Gly Thr Gly Gly Ala Thr Ala Asn Ser Ile Leu 34553460 3465 Gly Gln Leu Lys Ala Glu Leu Gly Asn Gln Trp Ile Ala Ala Thr3470 3475 3480 Thr Ala Cys Ala Thr Ala Thr Cys Ala Thr Thr Cys Thr GlyThr 3485 3490 3495 Gly Gly Thr Cys Thr Thr Ala Gly Ala Ala Cys Ala CysAla Thr 3500 3505 3510 Gly Cys Ala Gly Ala Gly Cys Thr Cys Gly Ala AlaGly Gly Ala 3515 3520 3525 Asn Tyr Ile Ser Phe Cys Gly Leu Arg Thr HisAla Glu Leu Glu 3530 3535 3540 Gly Ala Ala Cys Cys Thr Ala Gly Thr AlaAla Cys Thr Gly Ala 3545 3550 3555 Gly Cys Thr Thr Ala Thr Cys Thr AlaThr Gly Thr Cys Cys Ala 3560 3565 3570 Cys Ala Gly Cys Ala Ala Gly ThrThr Gly Thr Thr Ala Ala Thr 3575 3580 3585 Thr Gly Cys Thr Asn Leu ValThr Glu Leu Ile Tyr Val His Ser 3590 3595 3600 Lys Leu Leu Ile Ala GlyAla Thr Gly Ala Thr Ala Ala Cys Ala 3605 3610 3615 Cys Thr Gly Thr ThrAla Thr Thr Ala Thr Thr Gly Gly Cys Thr 3620 3625 3630 Cys Thr Gly CysCys Ala Ala Cys Ala Thr Ala Ala Ala Thr Gly 3635 3640 3645 Ala Cys CysGly Cys Ala Gly Cys Asp Asp Asn Thr Val Ile Ile 3650 3655 3660 Gly SerAla Asn Ile Asn Asp Arg Ser Ala Thr Gly Cys Thr Gly 3665 3670 3675 GlyGly Ala Ala Ala Gly Cys Gly Thr Gly Ala Cys Ala Gly Thr 3680 3685 3690Gly Ala Ala Ala Thr Gly Gly Cys Thr Gly Thr Cys Ala Thr Thr 3695 37003705 Gly Thr Gly Cys Ala Ala Gly Ala Thr Ala Cys Ala Met Leu Gly 37103715 3720 Lys Arg Asp Ser Glu Met Ala Val Ile Val Gln Asp Thr Gly Ala3725 3730 3735 Gly Ala Cys Thr Gly Thr Thr Cys Cys Thr Thr Cys Ala GlyThr 3740 3745 3750 Ala Ala Thr Gly Gly Ala Thr Gly Gly Ala Ala Ala AlaGly Ala 3755 3760 3765 Gly Thr Ala Cys Cys Ala Ala Gly Cys Thr Gly GlyCys Cys Gly 3770 3775 3780 Gly Glu Thr Val Pro Ser Val Met Asp Gly LysGlu Tyr Gln Ala 3785 3790 3795 Gly Arg Thr Thr Thr Gly Cys Cys Cys GlyAla Gly Gly Ala Cys 3800 3805 3810 Thr Thr Cys Gly Gly Cys Thr Ala CysAla Gly Thr Gly Cys Thr 3815 3820 3825 Thr Thr Ala Gly Gly Gly Thr ThrGly Thr Cys Cys Thr Thr Gly 3830 3835 3840 Gly Cys Thr Ala Thr Phe AlaArg Gly Leu Arg Leu Gln Cys Phe 3845 3850 3855 Arg Val Val Leu Gly TyrCys Thr Thr Gly Ala Thr Gly Ala Cys 3860 3865 3870 Cys Cys Ala Ala GlyThr Gly Ala Gly Gly Ala Cys Ala Thr Thr 3875 3880 3885 Cys Ala Gly GlyAla Thr Cys Cys Ala Gly Thr Gly Ala Gly Thr 3890 3895 3900 Gly Ala CysAla Ala Ala Thr Thr Cys Leu Asp Asp Pro Ser Glu 3905 3910 3915 Asp IleGln Asp Pro Val Ser Asp Lys Phe Thr Thr Cys Ala Ala 3920 3925 3930 GlyGly Ala Gly Gly Thr Gly Thr Gly Gly Gly Thr Thr Thr Cys 3935 3940 3945Ala Ala Cys Ala Gly Cys Ala Gly Cys Thr Cys Gly Ala Ala Ala 3950 39553960 Thr Gly Cys Thr Ala Cys Ala Ala Thr Thr Thr Ala Thr Phe Lys 39653970 3975 Glu Val Trp Val Ser Thr Ala Ala Arg Asn Ala Thr Ile Tyr Gly3980 3985 3990 Ala Cys Ala Ala Gly Gly Thr Thr Thr Thr Cys Cys Gly GlyThr 3995 4000 4005 Gly Cys Cys Thr Thr Cys Cys Cys Ala Ala Thr Gly AlaThr Gly 4010 4015 4020 Ala Ala Gly Thr Ala Cys Ala Cys Ala Ala Thr ThrThr Ala Ala 4025 4030 4035 Thr Thr Asp Lys Val Phe Arg Cys Leu Pro AsnAsp Glu Val His 4040 4045 4050 Asn Leu Ile Cys Ala Gly Cys Thr Gly AlaGly Ala Gly Ala Cys 4055 4060 4065 Thr Thr Thr Ala Thr Ala Ala Ala CysAla Ala Gly Cys Cys Cys 4070 4075 4080 Gly Thr Ala Thr Thr Ala Gly CysThr Ala Ala Gly Gly Ala Ala 4085 4090 4095 Gly Ala Thr Cys Cys Cys GlnLeu Arg Asp Phe Ile Asn Lys Pro 4100 4105 4110 Val Leu Ala Lys Glu AspPro Ala Thr Thr Cys Gly Ala Gly Cys 4115 4120 4125 Thr Gly Ala Gly GlyAla Gly Gly Ala Ala Cys Thr Gly Ala Ala 4130 4135 4140 Gly Ala Ala GlyAla Thr Cys Cys Gly Thr Gly Gly Ala Thr Thr 4145 4150 4155 Thr Thr ThrGly Gly Thr Gly Cys Ala Ala Ile Arg Ala Glu Glu 4160 4165 4170 Glu LeuLys Lys Ile Arg Gly Phe Leu Val Gln Thr Thr Cys Cys 4175 4180 4185 CysCys Thr Thr Thr Thr Ala Thr Thr Thr Cys Thr Thr Gly Thr 4190 4195 4200Cys Thr Gly Ala Ala Gly Ala Ala Ala Gly Cys Cys Thr Ala Cys 4205 42104215 Thr Gly Cys Cys Thr Thr Cys Thr Gly Thr Thr Gly Gly Gly Phe 42204225 4230 Pro Phe Tyr Phe Leu Ser Glu Glu Ser Leu Leu Pro Ser Val Gly4235 4240 4245 Ala Cys Cys Ala Ala Ala Gly Ala Gly Gly Cys Cys Ala ThrAla 4250 4255 4260 Gly Thr Gly Cys Cys Cys Ala Thr Gly Gly Ala Gly GlyThr Thr 4265 4270 4275 Thr Gly Gly Ala Cys Thr Thr Ala Ala Gly Ala GlyAla Thr Ala 4280 4285 4290 Thr Thr Lys Glu Ala Ile Val Pro Met Glu ValTrp Thr Thr Cys 4295 4300 4305 Ala Thr Thr Gly Gly Cys Ala Gly Cys ThrCys Ala Ala Ala Gly 4310 4315 4320 Ala Cys Thr Thr Cys Cys Ala Cys CysCys Thr Gly Gly Ala Gly 4325 4330 4335 Ala Cys Cys Ala Cys Ala Cys ThrGly Cys Ala Cys Ala Cys Ala 4340 4345 4350 Gly Thr Gly Ala Cys Thr ThrCys Cys Thr Gly Gly Gly Gly Ala 4355 4360 4365 Thr Gly Thr Cys Ala ThrAla Gly Cys Cys Ala Ala Ala Gly Cys 4370 4375 4380 Cys Ala Gly Gly CysCys Thr Gly Ala Cys Gly Cys Ala Thr Thr 4385 4390 4395 Cys Thr Cys GlyThr Ala Thr Cys Cys Ala Ala Cys Cys Cys Ala 4400 4405 4410 Ala Gly GlyAla Cys Cys Thr Thr Thr Thr Gly Gly Ala Ala Thr 4415 4420 4425 Gly AlaCys Thr Gly Gly Gly Gly Ala Gly Gly Gly Cys Thr Gly 4430 4435 4440 CysAla Gly Thr Cys Ala Cys Ala Thr Thr Gly Ala Thr Gly Thr 4445 4450 4455Ala Ala Gly Gly Ala Cys Thr Gly Thr Ala Ala Ala Cys Ala Thr 4460 44654470 Cys Ala Gly Cys Ala Ala Gly Ala Cys Thr Thr Thr Ala Thr Ala 44754480 4485 Ala Thr Thr Cys Cys Thr Thr Cys Thr Gly Cys Cys Thr Ala Ala4490 4495 4500 Cys Thr Thr Gly Thr Ala Ala Ala Ala Ala Gly Gly Gly GlyGly 4505 4510 4515 Cys Thr Gly Cys Ala Thr Thr Cys Thr Thr Gly Thr ThrGly Gly 4520 4525 4530 Thr Ala Gly Cys Ala Thr Gly Thr Ala Cys Thr CysThr Gly Thr 4535 4540 4545 Thr Gly Ala Gly Thr Ala Ala Ala Ala Cys AlaCys Ala Thr Ala 4550 4555 4560 Thr Thr Cys Ala Ala Ala Thr Thr Cys CysGly Cys Thr Cys Gly 4565 4570 4575 Thr Gly Cys Cys Gly Ala Ala Thr ThrCys 4580 4585 4 1036 PRT Homo sapiens 4 Met Ser Leu Lys Asn Glu Pro ArgVal Asn Thr Ser Ala Leu Gln Lys 1 5 10 15 Ile Ala Ala Asp Met Ser AsnIle Ile Glu Asn Leu Asp Thr Arg Glu 20 25 30 Leu His Phe Glu Gly Glu GluVal Asp Tyr Asp Val Ser Pro Ser Asp 35 40 45 Pro Lys Ile Gln Glu Val TyrIle Pro Phe Ser Ala Ile Tyr Asn Thr 50 55 60 Gln Gly Phe Lys Glu Pro AsnIle Gln Thr Tyr Leu Ser Gly Cys Pro 65 70 75 80 Ile Lys Ala Gln Val LeuGlu Val Glu Arg Phe Thr Ser Thr Thr Arg 85 90 95 Val Pro Ser Ile Asn LeuTyr Thr Ile Glu Leu Thr His Gly Glu Phe 100 105 110 Lys Trp Gln Val LysArg Lys Phe Lys His Phe Gln Glu Phe His Arg 115 120 125 Glu Leu Leu LysTyr Lys Ala Phe Ile Arg Ile Pro Ile Pro Thr Arg 130 135 140 Arg His ThrPhe Arg Arg Gln Asn Val Arg Glu Glu Pro Arg Glu Met 145 150 155 160 ProSer Leu Pro Arg Ser Ser Glu Asn Met Ile Arg Glu Glu Gln Phe 165 170 175Leu Gly Arg Arg Lys Gln Leu Glu Asp Tyr Leu Thr Lys Ile Leu Lys 180 185190 Met Pro Met Tyr Arg Asn Tyr His Ala Thr Thr Glu Phe Leu Asp Ile 195200 205 Ser Gln Leu Ser Phe Ile His Asp Leu Gly Pro Lys Gly Ile Glu Gly210 215 220 Met Ile Met Lys Arg Ser Gly Gly His Arg Ile Pro Gly Leu AsnCys 225 230 235 240 Cys Gly Gln Gly Arg Ala Cys Tyr Arg Trp Ser Lys ArgTrp Leu Ile 245 250 255 Val Lys Asp Ser Phe Leu Leu Tyr Met Lys Pro AspSer Gly Ala Ile 260 265 270 Ala Phe Val Leu Leu Val Asp Lys Glu Phe LysIle Lys Val Gly Lys 275 280 285 Lys Glu Thr Glu Thr Lys Tyr Gly Ile ArgIle Asp Asn Leu Ser Arg 290 295 300 Thr Leu Ile Leu Lys Cys Asn Ser TyrArg His Ala Arg Trp Trp Gly 305 310 315 320 Gly Ala Ile Glu Glu Phe IleGln Lys His Gly Thr Asn Phe Leu Lys 325 330 335 Asp His Arg Phe Gly SerTyr Ala Ala Ile Gln Glu Asn Ala Leu Ala 340 345 350 Lys Trp Tyr Val AsnAla Lys Gly Tyr Phe Glu Asp Val Ala Asn Ala 355 360 365 Met Glu Glu AlaAsn Glu Glu Ile Phe Ile Thr Asp Trp Trp Leu Ser 370 375 380 Pro Glu IlePhe Leu Lys Arg Pro Val Val Glu Gly Asn Arg Trp Arg 385 390 395 400 LeuAsp Cys Ile Leu Lys Arg Lys Ala Gln Gln Gly Val Arg Ile Phe 405 410 415Ile Met Leu Tyr Lys Glu Val Glu Leu Ala Leu Gly Ile Asn Ser Glu 420 425430 Tyr Thr Lys Arg Thr Leu Met Arg Leu His Pro Asn Ile Lys Val Met 435440 445 Arg His Pro Asp His Val Ser Ser Thr Val Tyr Leu Trp Ala His His450 455 460 Glu Lys Leu Val Ile Ile Asp Gln Ser Val Ala Phe Val Gly GlyIle 465 470 475 480 Asp Leu Ala Tyr Gly Arg Trp Asp Asp Asn Glu His ArgLeu Thr Asp 485 490 495 Val Gly Ser Val Lys Arg Val Thr Ser Gly Pro SerLeu Gly Ser Leu 500 505 510 Pro Pro Ala Ala Met Glu Ser Met Glu Ser LeuArg Leu Lys Asp Lys 515 520 525 Asn Glu Pro Val Gln Asn Leu Pro Ile GlnLys Ser Ile Asp Asp Val 530 535 540 Asp Ser Lys Leu Lys Gly Ile Gly LysPro Arg Lys Phe Ser Lys Phe 545 550 555 560 Ser Leu Tyr Lys Gln Leu HisArg His His Leu His Asp Ala Asp Ser 565 570 575 Ile Ser Ser Ile Asp SerThr Ser Asn Thr Gly Ser Ile Arg Ser Leu 580 585 590 Gln Thr Gly Val GlyGlu Leu His Gly Glu Thr Arg Phe Trp His Gly 595 600 605 Lys Asp Tyr CysAsn Phe Val Phe Lys Asp Trp Val Gln Leu Asp Lys 610 615 620 Pro Phe AlaAsp Phe Ile Asp Arg Tyr Ser Thr Pro Arg Met Pro Trp 625 630 635 640 HisAsp Ile Ala Ser Ala Val His Gly Lys Ala Ala Arg Asp Val Ala 645 650 655Arg His Phe Ile Gln Arg Trp Asn Phe Thr Lys Ile Met Lys Ser Lys 660 665670 Tyr Arg Ser Leu Ser Tyr Pro Phe Leu Leu Pro Lys Ser Gln Thr Thr 675680 685 Ala His Glu Leu Arg Tyr Gln Val Pro Gly Ser Val His Ala Asn Val690 695 700 Gln Leu Leu Arg Ser Ala Ala Asp Trp Ser Ala Gly Ile Lys TyrHis 705 710 715 720 Glu Glu Ser Ile His Ala Ala Tyr Val His Val Ile GluAsn Ser Arg 725 730 735 His Tyr Ile Tyr Ile Glu Asn Gln Phe Phe Ile SerCys Ala Asp Asp 740 745 750 Lys Val Val Phe Asn Lys Ile Gly Asp Ala IleAla Gln Arg Ile Leu 755 760 765 Lys Ala His Arg Glu Asn Gln Lys Tyr ArgVal Tyr Val Val Ile Pro 770 775 780 Leu Leu Pro Gly Phe Glu Gly Asp IleSer Thr Gly Gly Gly Asn Ala 785 790 795 800 Leu Gln Ala Ile Met His PheAsn Tyr Arg Thr Met Cys Arg Gly Glu 805 810 815 Asn Ser Ile Leu Gly GlnLeu Lys Ala Glu Leu Gly Asn Gln Trp Ile 820 825 830 Asn Tyr Ile Ser PheCys Gly Leu Arg Thr His Ala Glu Leu Glu Gly 835 840 845 Asn Leu Val ThrGlu Leu Ile Tyr Val His Ser Lys Leu Leu Ile Ala 850 855 860 Asp Asp AsnThr Val Ile Ile Gly Ser Ala Asn Ile Asn Asp Arg Ser 865 870 875 880 MetLeu Gly Lys Arg Asp Ser Glu Met Ala Val Ile Val Gln Asp Thr 885 890 895Glu Thr Val Pro Ser Val Met Asp Gly Lys Glu Tyr Gln Ala Gly Arg 900 905910 Phe Ala Arg Gly Leu Arg Leu Gln Cys Phe Arg Val Val Leu Gly Tyr 915920 925 Leu Asp Asp Pro Ser Glu Asp Ile Gln Asp Pro Val Ser Asp Lys Phe930 935 940 Phe Lys Glu Val Trp Val Ser Thr Ala Ala Arg Asn Ala Thr IleTyr 945 950 955 960 Asp Lys Val Phe Arg Cys Leu Pro Asn Asp Glu Val HisAsn Leu Ile 965 970 975 Gln Leu Arg Asp Phe Ile Asn Lys Pro Val Leu AlaLys Glu Asp Pro 980 985 990 Ile Arg Ala Glu Glu Glu Leu Lys Lys Ile ArgGly Phe Leu Val Gln 995 1000 1005 Phe Pro Phe Tyr Phe Leu Ser Glu GluSer Leu Leu Pro Ser Val 1010 1015 1020 Gly Thr Lys Glu Ala Ile Val ProMet Glu Val Trp Thr 1025 1030 1035 5 3108 DNA Homo sapiens 5 atgtcactgaaaaacgagcc acgggtaaat acctctgcac tgcagaaaat tgctgctgac 60 atgagtaatatcatagaaaa tctggacacg cgggaactcc actttgaggg agaggaggta 120 gactacgacgtgtctcccag cgatcccaag atacaagaag tgtatatccc tttctctgct 180 atttataacactcaaggatt taaggagcct aatatacaga cgtatctctc cggctgtcca 240 ataaaagcacaagttctgga agtggaacgc ttcacatcta caacaagggt accaagtatt 300 aatctttacactattgaatt aacacatggg gaatttaaat ggcaagttaa gaggaaattc 360 aagcattttcaagaatttca cagagagctg ctcaagtaca aagcctttat ccgcatcccc 420 attcccactagaagacacac gtttaggagg caaaacgtca gagaggagcc tcgagagatg 480 cccagtttgccccgttcatc tgaaaacatg ataagagaag aacaattcct tggtagaaga 540 aaacaactggaagattactt gacaaagata ctaaaaatgc ccatgtatag aaactatcat 600 gccacaacagagtttcttga tataagccag ctgtctttca tccatgattt gggaccaaag 660 ggcatagaaggtatgataat gaaaagatct ggaggacaca gaataccagg cttgaattgc 720 tgtggtcagggaagagcctg ctacagatgg tcaaaaagat ggttaatagt gaaagattcc 780 tttttattgtatatgaaacc agacagcggt gccattgcct tcgtcctgct ggtagacaaa 840 gaattcaaaattaaggtggg gaagaaggag acagaaacga aatatggaat ccgaattgat 900 aatctttcaaggacacttat tttaaaatgc aacagctata gacatgctcg gtggtgggga 960 ggggctatagaagaattcat ccagaaacat ggcaccaact ttctcaaaga tcatcgattt 1020 gggtcatatgctgctatcca agagaatgct ttagctaaat ggtatgttaa tgccaaagga 1080 tattttgaagatgtggcaaa tgcaatggaa gaggcaaatg aagagatttt tatcacagac 1140 tggtggctgagtccagaaat cttcctgaaa cgcccagtgg ttgagggaaa tcgttggagg 1200 ttggactgcattcttaaacg aaaagcacaa caaggagtga ggatcttcat aatgctctac 1260 aaagaggtggaactcgctct tggcatcaat agtgaataca ccaagaggac tttgatgcgt 1320 ctacatcccaacataaaggt gatgagacac ccggatcatg tgtcatccac cgtctatttg 1380 tgggctcaccatgagaagct tgtcatcatt gaccaatcgg tggcctttgt gggagggatt 1440 gacctggcctatggaaggtg ggacgacaat gagcacagac tcacagacgt gggcagtgtg 1500 aagcgggtcacttcaggacc gtctctgggt tccctcccac ctgccgcaat ggagtctatg 1560 gaatccttaagactcaaaga taaaaatgag cctgttcaaa acctacccat ccagaagagt 1620 attgatgatgtggattcaaa actgaaagga ataggaaagc caagaaagtt ctccaaattt 1680 agtctctacaagcagctcca caggcaccac ctgcacgacg cagatagcat cagcagcatt 1740 gacagcacctccaataccgg gtccatccgt agtttacaga caggtgtggg agagctgcat 1800 ggggaaaccagattctggca tggaaaggac tactgcaatt tcgtcttcaa agactgggtt 1860 caacttgataaaccttttgc tgatttcatt gacaggtact ccacgccccg gatgccctgg 1920 catgacattgcctctgcagt ccacgggaag gcggctcgtg atgtggcacg tcacttcatc 1980 cagcgctggaacttcacaaa aattatgaaa tcaaaatatc ggtccctttc ttatcctttt 2040 ctgcttccaaagtctcaaac aacagcccat gagttgagat atcaagtgcc tgggtctgtc 2100 catgctaacgtacagttgct ccgctctgct gctgattggt ctgctggtat aaagtaccat 2160 gaagagtccatccacgccgc ttacgtccat gtgatagaga acagcaggca ctatatctat 2220 atcgaaaaccagtttttcat aagctgtgct gatgacaaag ttgtgttcaa caagataggc 2280 gatgccattgcccagaggat cctgaaagct cacagggaaa accagaaata ccgggtatat 2340 gtcgtgataccacttctgcc agggttcgaa ggagacattt caaccggcgg aggaaatgct 2400 ctacaggcaatcatgcactt caactacaga accatgtgca gaggagaaaa ttccatcctt 2460 ggacagttaaaagcagagct tggtaatcag tggataaatt acatatcatt ctgtggtctt 2520 agaacacatgcagagctcga aggaaaccta gtaactgagc ttatctatgt ccacagcaag 2580 ttgttaattgctgatgataa cactgttatt attggctctg ccaacataaa tgaccgcagc 2640 atgctgggaaagcgtgacag tgaaatggct gtcattgtgc aagatacaga gactgttcct 2700 tcagtaatggatggaaaaga gtaccaagct ggccggtttg cccgaggact tcggctacag 2760 tgctttagggttgtccttgg ctatcttgat gacccaagtg aggacattca ggatccagtg 2820 agtgacaaattcttcaagga ggtgtgggtt tcaacagcag ctcgaaatgc tacaatttat 2880 gacaaggttttccggtgcct tcccaatgat gaagtacaca atttaattca gctgagagac 2940 tttataaacaagcccgtatt agctaaggaa gatcccattc gagctgagga ggaactgaag 3000 aagatccgtggatttttggt gcaattcccc ttttatttct tgtctgaaga aagcctactg 3060 ccttctgttgggaccaaaga ggccatagtg cccatggagg tttggact 3108 6 932 PRT Homo sapiens 6Met Thr Val Thr Gln Lys Asn Leu Phe Pro Tyr Gly Asp Tyr Leu Asn 1 5 1015 Ser Ser Gln Leu His Met Glu Pro Asp Glu Val Asp Thr Leu Arg Glu 20 2530 Gly Glu Asp Pro Ala Asp Arg Met His Pro Tyr Leu Ala Ile Tyr Asp 35 4045 Leu Gln Pro Leu Lys Ala His Pro Leu Val Phe Ala Pro Gly Val Pro 50 5560 Val Ile Ala Gln Val Val Gly Thr Glu Arg Tyr Thr Ser Gly Ser Lys 65 7075 80 Val Gly Thr Cys Thr Leu Tyr Ser Val Arg Leu Thr His Gly Asp Phe 8590 95 Thr Trp Thr Thr Lys Lys Lys Phe Arg His Phe Gln Glu Leu His Arg100 105 110 Asp Leu Gln Arg His Lys Val Leu Met Ser Leu Leu Pro Leu AlaArg 115 120 125 Phe Ala Val Thr His Ser Pro Ala Arg Glu Ala Ala Ala GluAsp Ile 130 135 140 Pro Ser Leu Pro Arg Gly Gly Ser Glu Gly Ser Ala ArgHis Thr Ala 145 150 155 160 Ser Lys Gln Lys Tyr Leu Glu Asn Tyr Leu AsnArg Leu Leu Thr Met 165 170 175 Ser Phe Tyr Arg Asn Tyr His Ala Met ThrGlu Phe Leu Glu Val Ser 180 185 190 Gln Leu Ser Phe Ile Pro Asp Leu GlySer Lys Gly Leu Glu Gly Val 195 200 205 Ile Arg Lys Arg Ser Gly Gly HisArg Val Pro Gly Phe Thr Phe Cys 210 215 220 Gly Arg Asp Gln Val Cys TyrArg Trp Ser Lys Arg Trp Leu Val Val 225 230 235 240 Lys Asp Ser Phe LeuLeu Tyr Met Arg Pro Glu Thr Gly Ala Ile Ser 245 250 255 Phe Val Gln LeuPhe Asp Pro Gly Phe Glu Val Gln Val Gly Lys Arg 260 265 270 Ser Thr GluThr Arg Tyr Gly Val Arg Ile Asp Thr Ser His Arg Ser 275 280 285 Leu IleLeu Lys Cys Ser Ser Tyr Arg Gln Ala Arg Trp Trp Gly Gln 290 295 300 GluIle Thr Glu Leu Ala Gln Gly Ser Gly Arg Asp Phe Leu Gln Leu 305 310 315320 His Gln His Asp Ser Tyr Ala Pro Pro Arg Pro Gly Thr Leu Ala Arg 325330 335 Trp Phe Val Asn Gly Ala Gly Tyr Phe Ala Ala Val Ala Asp Ala Ile340 345 350 Leu Arg Ala Gln Glu Glu Ile Phe Ile Thr Asp Trp Trp Leu SerPro 355 360 365 Glu Ile Tyr Leu Lys Arg Pro Ala His Ser Asp Asp Trp ArgLeu Asp 370 375 380 Ile Met Leu Lys Arg Lys Ala Glu Glu Gly Val Arg ValSer Ile Leu 385 390 395 400 Leu Phe Lys Glu Val Glu Leu Ala Leu Gly IleAsn Ser Gly Tyr Ser 405 410 415 Lys Arg Thr Leu Met Leu Leu His Pro AsnIle Lys Val Met Arg His 420 425 430 Pro Asp Leu Val Thr Leu Trp Ala HisHis Glu Lys Leu Leu Val Val 435 440 445 Asp Gln Val Val Ala Phe Leu GlyGly Leu Asp Leu Ala Phe Gly Arg 450 455 460 Trp Asp Asp Val Gln Tyr ArgLeu Thr Asp Leu Gly Asp Pro Ser Glu 465 470 475 480 Pro Val His Leu GlnThr Pro Thr Leu Gly Ser Asp Pro Ala Ala Thr 485 490 495 Pro Asp Leu SerHis Asn Gln Phe Phe Trp Leu Gly Lys Asp Tyr Ser 500 505 510 Asn Leu IleThr Lys Asp Trp Val Gln Leu Asp Arg Pro Phe Glu Asp 515 520 525 Phe IleAsp Arg Glu Thr Thr Pro Arg Met Pro Trp Arg Asp Val Gly 530 535 540 ValVal Val His Gly Val Ala Ala Arg Asp Leu Ala Arg His Phe Ile 545 550 555560 Gln Arg Trp Asn Phe Thr Lys Thr Thr Lys Ala Arg Tyr Lys Thr Pro 565570 575 Leu Tyr Pro Tyr Leu Leu Pro Lys Ser Thr Ser Thr Ala Asn Asn Leu580 585 590 Pro Phe Met Ile Pro Gly Gly Gln Cys Ala Thr Val Gln Val LeuArg 595 600 605 Ser Val Asp Arg Trp Ser Ala Gly Thr Leu Glu Asn Ser IleLeu Asn 610 615 620 Ala Tyr Leu His Thr Ile Arg Glu Ser Gln His Phe LeuTyr Ile Glu 625 630 635 640 Asn Gln Phe Phe Ile Ser Cys Ser Asp Gly ArgThr Val Leu Asn Lys 645 650 655 Val Gly Asp Glu Ile Val Asp Arg Ile LeuLys Ala His Glu Gln Gly 660 665 670 Gln Cys Phe Arg Val Tyr Leu Leu LeuPro Leu Leu Pro Gly Phe Glu 675 680 685 Gly Asp Ile Ser Thr Gly Gly GlyAsn Ser Ile Gln Ala Ile Leu His 690 695 700 Phe Thr Tyr Arg Thr Leu CysArg Gly Glu His Ser Ile Leu His Arg 705 710 715 720 Leu Lys Ala Ala MetGly Thr Ala Trp Arg Asp Tyr Met Ser Ile Cys 725 730 735 Gly Leu Arg ThrHis Gly Glu Leu Gly Gly His Pro Ile Ser Glu Leu 740 745 750 Ile Tyr IleHis Ser Lys Met Leu Ile Ala Asp Asp Arg Thr Val Ile 755 760 765 Ile GlySer Ala Asn Ile Asn Asp Arg Ser Leu Leu Gly Lys Arg Asp 770 775 780 SerGlu Leu Ala Ile Leu Ile Lys Asp Thr Glu Met Glu Pro Ser Leu 785 790 795800 Met Asp Gly Val Glu Tyr Gln Ala Gly Arg Phe Ala Leu Ser Leu Arg 805810 815 Gly Arg Cys Phe Ser Val Ile Leu Gly Ala Asn Thr Trp Pro Asp Leu820 825 830 Asp Leu Arg Asp Pro Val Cys Asp Asp Phe Phe Gln Leu Trp GlnGlu 835 840 845 Thr Ala Glu Asn Asn Ala Thr Ile Tyr Glu Gln Ile Phe ArgCys Leu 850 855 860 Pro Ser Asn Ala Thr Arg Ser Leu Arg Leu Ser Gly SerMet Trp Leu 865 870 875 880 Trp Ser Pro Trp Leu Gln Ser Ala Phe Leu AlaGln Ser Glu Leu Ala 885 890 895 His Ile Gln Gly His Leu Val His Phe ProLeu Lys Phe Leu Glu Asp 900 905 910 Glu Ser Leu Leu Pro Pro Leu Gly SerLys Glu Gly Met Ile Pro Leu 915 920 925 Glu Val Trp Thr 930 7 22 DNAHomo sapiens 7 tccatccagg ccattctgca ct 22 8 22 DNA Homo sapiens 8cgttgctctc agccatgtct tg 22

What is claimed is:
 1. An isolated DNA sequence that codes on expressionfor a PLD polypeptide.
 2. An isolated DNA sequence that codes onexpression for a PLD1a polypeptide.
 3. An isolated DNA sequence thatcodes on expression for a PLD1b polypeptide.
 4. An isolated DNA sequencethat codes on expression for a PLD2 polypeptide.
 5. An isolated DNAsequence selected from the group consisting of: a) the DNA correspondingto the nucleotide sequence of SEQ ID NO:1; b) the DNA corresponding tothe nucleotide sequence of SEQ ID NO:3; c) the DNA corresponding to thenucleotide sequence of SEQ ID NO:4; d) the DNA corresponding to thenucleotide sequence of SEQ ID NO:6; e) the DNA corresponding to thenucleotide sequence of SEQ ID NO:7; f) the DNA corresponding to thenucleotide sequence of SEQ ID NO:9; and g) a DNA capable of hybridizingunder stringent conditions, or which would be capable of hybridizingunder said conditions but for the degeneracy of the genetic code, to asequence corresponding to the sequence of SEQ ID NO:1, or SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.
 6. A vectorcomprising a DNA sequence of claim 1, 2, 3, 4 or
 5. 7. A celltransformed with a DNA sequence of claim 1, 2, 3, 4 or 5, said DNAsequence being arranged in operative association with an expressioncontrol sequence capable of directing replication and expression of saidDNA sequence.
 8. A cell according to claim 7 wherein said cell is amammalian cell or an insect cell.
 9. A process for producing a PLDprotein comprising culturing a cell of claim 7 in a suitable culturemedium and isolating said PLD protein from said cell.
 10. A method foridentifying mediators of PLD comprising the step of: a) transfecting acell line with an expression vector comprising nucleic acid sequencesencoding a PLD protein; b) culturing said cell line in a culture medium,whereby said PLD protein is expressed stably; c) adding an effectiveamount of a compound to said culture medium, sufficient to cause adetectable loss in the catalytic activity of PLD; and d) detecting saidloss in catalytic activity.
 11. A method of claim 10 wherein saiddetection is by an immunoassay.
 12. A method of claim 10 wherein saidPLD is labeled.
 13. A pharmaceutical composition useful in the treatmentof wound healing comprising a therapeutically effective amount of a PLDmediator identified using the method of claim 10 in a pharmaceuticallyacceptable vehicle.
 14. A pharmaceutical composition useful in thetreatment of autoimmune diseases comprising a therapeutically effectiveamount of a PLD mediator identified using the method of claim 10 in apharmaceutically acceptable vehicle.
 15. A pharmaceutical compositionuseful in the treatment of inflammatory diseases comprising atherapeutically effective amount of a PLD mediator identified using themethod of claim 10 in a pharmaceutically acceptable vehicle.
 16. Apolypeptide substantially free of association with other polypeptidesand comprising an amino acid sequence selected from the group consistingof a) the amino acid sequence of SEQ ID NO:2; b) the amino acid sequenceof SEQ ID NO:5; and c. the amino acid sequence of SEQ ID NO:8.
 17. Apolypeptide encoded by a DNA sequence of claim
 5. 18. A polypeptidesubstantially free of association with other polypeptides and comprisingan enzyme of mammalian origin having a phosphatidylcholine-specificphospholipase D activity and containing at least two copies of the aminoacid motif HXKXXXXD.
 19. A polypeptide substantially free of associationwith other polypeptides and comprising a PLD polypeptide that: a) isperinuclear membrane associated, b) requires PI(4,5)P2 for in vitroactivity and c) is activated by one or more G-proteins.
 20. Apolypeptide substantially free of association with other polypeptidesand comprising a PLD polypeptide that: a) is plasma membrane associated,b) activates cytoskelatal reorganization pathways, c) requires PI(4,5)P2for in vitro activity and d) does not require Rac1, cdc42, RhoA, PKC orARF1 for activation.
 21. A composition comprising a PLD1 or PLD2 ofpolypeptide of claim 18, claim 19 or claim 20 in combination with aG-protein.
 22. A composition of claim 21 wherein said G-protein isselected from the group consisting of ADP-ribosylation factor 1, RhoA,Rac1 and cdc42.